JP7425946B2 - Adhesion inhibitors for proteins, cells or microorganisms, and their uses - Google Patents

Description

The present invention relates to an adhesion inhibitor for proteins, cells, or microorganisms, as well as a blocking agent for biochemical analysis containing the adhesion inhibitor, a coating agent for inhibiting biofilm formation, a treatment agent for cell culture equipment, a protein stabilizer, and a cell culture medium. Regarding additives.

In the field of biochemical analysis, methods for detecting and visualizing DNA and proteins using specific adsorption through antigen-antibody reactions are widely known as Southern blotting, Western blotting, ELISA, immunostaining, and the like. Antibodies also cause nonspecific adsorption reactions with other than target proteins. In order to prevent such non-specific adsorption, pretreatment is performed such that the surface to be detected or measured is coated with a blocking agent that prevents non-specific adsorption but does not interfere with specific adsorption.
As blocking agents, proteins derived from living organisms such as normal serum, bovine serum albumin, gelatin, and skim milk are known (Patent Document 1, Non-Patent Document 1). Also known as blocking agents include a surfactant called TWEEN20, hydroxyalkyl cellulose, polyvinyl alcohol, and copolymers of (meth)acryloylmorpholine and other monomers (Patent Documents 2, 3, 4). Further, blocking agents using copolymers having phosphorylcholine groups in their side chains have also been studied (Patent Document 5, Non-Patent Document 2).
However, biologically-derived proteins such as those disclosed in Patent Document 1 and Non-Patent Document 1 have a potential problem that variations in performance tend to occur. By the way, the blocking agent specifically adsorbs the staining agent to the protein that is the target of staining and does not interfere with the color development, and also covers proteins other than the target and prevents the staining agent from adsorbing to proteins other than the target. It is required that the color does not develop without coloring. However, the surfactants disclosed in Patent Documents 2 to 4 prevent color development due to specific adsorption of staining agents to target proteins, cannot sufficiently cover proteins other than the target, and stain proteins other than the target as well. There are problems such as adsorption of agents and color development. Furthermore, the copolymer disclosed in Patent Document 5 has problems in productivity, such as requiring multiple reactions and accompanying purification during synthesis of raw material monomers.

Biofilm is also referred to as biological film or slime, and generally occurs in water systems when microorganisms such as bacteria and mold adhere to and multiply on the surface of materials, producing polymeric substances such as polysaccharides and proteins from within the microbial cells. Refers to something that has formed a body. Comparing before and after a biofilm is formed, once a biofilm is formed, it shows significantly high resistance to cleaning/removal, antibiotics, drugs, heat, drying, etc. As a result, hazards caused by the attached and multiplied microorganisms occur, causing problems in various industrial fields.
For example, bacteria adhere to the inside of a medical device such as a catheter and form a biofilm, causing a blockage and making it impossible to perform the desired treatment. In addition, the biofilm may fall off, allowing bacterial aggregates to enter the body and cause serious illness. When a biofilm forms inside the pipes of a food plant, it not only peels off and leads to foreign matter getting into the product, but also causes food poisoning due to toxins derived from microorganisms. Furthermore, biofilm formation on metal surfaces causes metal corrosion and accelerates the deterioration of equipment. Furthermore, if a biofilm is formed on the inner surface of the aquarium, it will have a negative impact on the organisms in the aquarium.
As described above, there is a need to suppress the formation of biofilms, and various studies have been conducted to suppress the formation of biofilms.
Patent Document 6 discloses a biofilm treatment agent containing a low molecular weight amine oxide surfactant in a biofilm disintegrating agent mainly composed of an alkali inorganic salt. Techniques for cleaning with activators are disclosed. However, once a biofilm is formed, it is difficult to remove it, and the cleaning work imposes a heavy labor burden. Furthermore, since a large amount of water is used during cleaning, there are problems with treatment and environmental pollution. Patent Document 7 discloses a method for inhibiting biofilm formation characterized by a polymer compound derived from a vinyl monomer having one or more groups selected from amino groups and quaternary ammonium groups and having an anionic group. A method is disclosed in which the polymer compound exhibits microbial adhesion prevention, sterilization, and antibacterial effects, thereby suppressing biofilm formation. However, this polymer compound has poor water resistance and insufficient ability to inhibit biofilm formation.

In addition, evaluations using cells are widely performed in fields such as drug discovery of pharmaceuticals and agricultural chemicals, food testing, diagnosis, and genetic engineering. Conventionally, when evaluating cell functions and responses, cells are cultured in a culture medium placed in a petri dish, etc., and used in various experiments. However, when investigating the reactions caused by a large number of drugs and introduced substances, there is a need for an evaluation method that can efficiently analyze a variety of specific cell reactions in a short period of time and obtain results using fewer samples. Ta. Under these circumstances, attempts to observe or measure the chemical reactions of cells held on a substrate such as glass using a cell culture device called a microchip have the advantage of simplifying operations and shortening analysis time through automation. This is expected to be the case, and it has become popular in recent years. The advantage of a system using a microchip is that it reduces the amount of samples and reagents used and the amount of waste fluid discharged, and it is thought that it will be possible to realize a space-saving, portable, and inexpensive system. For example, in Non-Patent Document 3, there is also a study in which the functions and responses of cells are evaluated using a microchip in which cells are attached to the wall surface of a flow channel, taking advantage of the above-mentioned advantages.
However, microchips using cells have problems in that cells and proteins derived from biological samples adhere to the surface of the device, leading to clogging and a decrease in analysis accuracy and sensitivity. To solve this problem, for example, Patent Document 8 reports a microchannel device coated with a cell adhesion inhibitor containing a polymer having a repeating unit having a sulfinyl group in its side chain as an active ingredient. .

In addition, the higher-order structure of proteins is maintained by non-covalent weak polar interactions such as van der Waals interactions, hydrogen bonds, and electrostatic interactions within the polypeptide chain; Various functions are expressed by These bonds change depending on various external factors (temperature, pH, salt concentration, concentration of the protein itself, surfactants, denaturants, divalent metals, coexistence of proteases), and in some cases the structure of the protein may be destroyed. The original function is lost. Therefore, protein stability can be translated into the ability to maintain a functional higher-order structure.
For this reason, it is necessary to stabilize proteins in an aqueous solution, and it is necessary to optimize storage conditions, such as storing proteins at a specified temperature in a buffer solution, and to use stabilizers (other proteins such as albumin, glycerose, polyethylene, etc.). Polyhydric alcohols such as glycol and sucrose, amino acids such as glutamic acid and lysine, reducing agents such as glutathione and dithiothreitol, metal chelating agents such as EDTA, organic acid salts such as citric acid, heavy metal salts, substrates, and coenzymes). Their use alone or in combination is being considered (Non-Patent Document 4). In particular, as stabilizers for synthetic compounds, for example, Patent Document 9 describes a polymer having a phosphorylcholine group, Patent Document 10 describes a polymer compound having a polyethylene glycol moiety, and Patent Document 11 describes a low-molecular amine oxide compound. Methods of use are disclosed.
However, these methods may not have satisfactory retention rates or stability periods depending on the type of protein, and further improvements are required.

In addition, animal cells used in fields such as efficacy and toxicity evaluation of chemical substances and pharmaceuticals, mass production of useful substances such as antibodies, and regenerative medicine are stored in culture containers such as petri dishes, multiwell plates, and bag-like containers. Often cultivated. Cells in the culture container, for example, adhere to the surface of the culture container and grow two-dimensionally by spreading on the culture surface of the culture container. However, because the environment of two-dimensionally proliferated cells is very different from that in the living body, they may not express the morphology and function that they would have in the living body, so it is more advantageous to form three-dimensional cell aggregates to maintain their functions. It is said that Therefore, a technique for forming cell aggregates using a culture container to which cells are difficult to adhere has been disclosed. For example, Non-Patent Document 5 reports that hepatocytes form spheroids in a culture device coated with hydrophilic polyhydroxyethyl methacrylate. Further, Patent Document 12 discloses a culture vessel coated with a copolymer of 2-methacryloyloxyethylphosphorylcholine and butyl methacrylate.
However, with the techniques disclosed in Non-Patent Document 5 and Patent Document 12, it takes time and cost to treat the surface of the culture container with a coating agent. In addition, high voltage is applied to the polystyrene surface in an atmosphere of reduced pressure air, argon gas, etc. to generate plasma and generate radicals on the surface, and the radicals are caused to contact react with water vapor and oxygen, forming hydroxyl groups and carboxyl groups. Methods of generating hydrophilic functional groups have also been used, but with these methods, the polystyrene surface gradually changes to hydrophobicity after plasma treatment, making it impossible to maintain a hydrophilic surface state. There was a problem that dimensional cell aggregates could not be formed.

In addition, techniques for efficiently culturing animal cells in large quantities have been applied to the production of useful physiologically active substances such as monoclonal antibodies. A culture medium to which approximately 20% animal serum such as fetal bovine serum (FBS) is added is generally used. However, adding serum to the medium increases the cost of the medium and has disadvantages such as making it difficult to isolate the target physiologically active substance from the culture solution containing serum-derived proteins. Additionally, in many cases, serum quality varies between lots. Therefore, before using serum, it is necessary to perform a thorough lot check and select a usable serum, but selecting this serum requires a lot of effort.
In order to solve the above problems, studies have been conducted on media that do not use serum (serum-free media).
For example, Patent Document 13 discloses that by adding glycylglycine to a culture medium, animal cells can be made to produce useful substances at high concentrations. Further, Patent Document 14 discloses that physiologically active substances can be efficiently produced when cells are cultured in a medium containing a polymer having a structure similar to phospholipids. Further, Patent Document 15 discloses that a culture medium containing hydrophobic D-amino acids promotes cell proliferation and increases the production amount of useful substances.
However, most conventional serum-free media are not as satisfactory as serum-containing media in terms of proliferation, survival, and antibody productivity of antibody-producing cells. Further, in culturing antibody-producing cells, there is no knowledge regarding the effect of using a vinyl polymer having an amine oxide group as a medium component and having a mass average molecular weight of 2,000 or more as a medium additive.

International Publication No. 2016/052690 Japanese Patent Application Publication No. 2004-219111 Japanese Patent Application Publication No. 04-019561 Japanese Patent Application Publication No. 2008-209114 Japanese Patent Application Publication No. 07-083923 Japanese Patent Application Publication No. 2008-156389 Japanese Patent Application Publication No. 2010-163429 International Publication No. 2013/099901 Japanese Patent Application Publication No. 10-45794 Japanese Patent Application Publication No. 10-237094 Special Publication No. 2007-509164 International Publication No. 2005/001019 Japanese Patent Application Publication No. 7-23780 Japanese Unexamined Patent Publication No. 4-304882 JP2015-027265A

“Watanabe/Nakane Enzyme-Antibody Method” Published by Interdisciplinary Planning Co., Ltd. 2002 Polymer Papers Vol. 35 No. 7 423 (1978) Analytical Chemistry 2005:77, p. 2125-2131 New Biochemistry Experiment Course 1 Protein I Separation/Purification/Properties Chapter 16 EXPERIMENTAL CELL RESEARCH, 200, 326-332 (1992)

In view of the above circumstances, an object of the present invention is to provide an adhesion inhibitor for proteins, cells, or microorganisms that exhibits an excellent ability to inhibit adhesion of proteins, cells, or microorganisms, and various uses containing the adhesion inhibitor. It is in.

In addition, the present invention has the ability to not interfere with color development due to specific adsorption of the stain to the target protein, sufficiently cover proteins other than the target, prevent non-specific adsorption of the stain to proteins other than the target, and prevent color development. The purpose of the present invention is to provide a blocking agent for biochemical analysis that can stably exhibit the performance of biochemical analysis. That is, the present invention has the effect of sufficiently covering proteins other than the target without hindering color development due to specific adsorption of the stain to the target protein, and preventing non-specific adsorption of the stain to proteins other than the target, thereby preventing color development. The purpose of the present invention is to provide a blocking agent for biochemical analysis that exhibits stable performance.

Another object of the present invention is to provide a biofilm formation-inhibiting coating agent and a coating film made of the coating agent, which is excellent in safety, coatability, and water resistance, and which enables long-term inhibition of biofilm formation. An object of the present invention is to provide a biofilm formation-inhibiting laminate.

The present invention also provides a cell culture equipment treatment agent that suppresses protein adsorption and exhibits an excellent cell adhesion prevention effect, and a cell culture equipment treatment agent that has a cell adhesion prevention film made of the cell culture equipment treatment agent and is suitable for cell mass production. The present invention aims to provide a cell culture device, a medical device equipped with the device, and a method for manufacturing the cell culture device.

Another object of the present invention is that by using a vinyl polymer (A) that has an amine oxide group and a mass average molecular weight of 2,000 or more, stable storage can be achieved without impairing protein functions. , to provide a protein stabilizer. Another object of the present invention is to provide a method for stabilizing a protein, which allows stable storage without impairing the function of the protein by allowing the protein stabilizer and the protein to coexist.

Another object of the present invention is to provide a cell culture medium additive, a cell culture medium containing the additive, and a cell culture medium additive for producing a three-dimensional cell aggregate with a good proportion of viable cells without increasing cost or labor. The object of the present invention is to provide a culture medium and a method for producing cell aggregates using the additive.

The present invention also aims to provide an antibody-producing cell culture medium additive, a method for culturing antibody-producing cells using the additive, and a method for culturing antibody-producing cells to increase antibody productivity without increasing cost or labor. An object of the present invention is to provide a culture medium and a method for producing antibodies.

That is, the present invention relates to the following [1] to [26].

[1] An adhesion inhibitor for proteins, cells, or microorganisms, comprising a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 2,000 or more.

[2] The protein, cell or microorganism adhesion inhibitor according to [1], wherein the vinyl polymer (A) has at least one of the structures represented by the following general formulas 1 to 3.

[In general formulas 1 to 3,
X is a divalent bonding group,
y is 0 or 1,
R ¹ is an alkylene group having 1 to 6 carbon atoms,
R ² and R ³ are each independently an alkyl group having 1 to 4 carbon atoms,
R ⁴ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Four of R ⁵ to R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ⁵ to R ⁹ is a bond with the main chain of the vinyl polymer (A). represents the bond position,
* represents the bonding position with the main chain of the vinyl polymer (A). ]

[3] The vinyl polymer (A) is a reaction product of a polymer obtained by polymerizing a monomer having an ethylenically unsaturated group and an oxidizing agent,
[1] or [2], wherein the monomer having an ethylenically unsaturated group contains as an essential component a monomer (a) having one ethylenically unsaturated group and a tertiary amino group in one molecule. Adhesion inhibitor for proteins, cells or microorganisms.

[4] In [3], the monomer having an ethylenically unsaturated group contains at least a monomer (a) having a tertiary amino group and a monomer (c) having an alkyl group having 4 to 18 carbon atoms. An adhesion inhibitor for the proteins, cells or microorganisms described above.

[5] The adhesion inhibitor for proteins, cells, or microorganisms according to any one of [1] to [4] is included, and the vinyl polymer (A) in the adhesion inhibitor contains 1 to 10 mmol of amine oxide groups. A blocking agent for biochemical analysis, which is a vinyl polymer (A) having /g.

[6] The blocking agent for biochemical analysis according to [5], wherein the vinyl polymer (A) is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer.

[7] After bringing the blocking agent for biochemical analysis according to [5] or [6] into contact with the pathological tissue fixed on the surface of the base material, adsorbing the staining antibody to the antigen in the pathological tissue, An immunostaining method for detecting the antigen.

[8] An amine oxide group-containing vinyl polymer (A') comprising a thiol group-containing compound (x) and a vinyl monomer, the amine oxide group-containing vinyl polymer (A') having the following general formula 1. An amine oxide group-containing vinyl polymer (A') containing at least one of the amine oxide structures represented by .

[In general formulas 1 to 3,
X is a divalent bonding group,
y is 0 or 1,
R ¹ is an alkylene group having 1 to 6 carbon atoms,
R ² and R ³ are each independently an alkyl group having 1 to 4 carbon atoms,
R ⁴ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Four of R ⁵ to R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ⁵ to R ⁹ is connected to the main chain of the vinyl polymer (A'). represents the bond position of
* represents the bonding position with the main chain of the vinyl polymer (A'). ]

[9] Contains the adhesion inhibitor for proteins, cells, or microorganisms according to any one of [1] to [4], wherein the adhesion inhibitor has a mass average molecular weight of 2,000 to 10,000,000. A biofilm formation inhibiting coating agent containing a vinyl polymer (A).

[10] The biofilm formation-inhibiting coating agent according to [9], wherein the vinyl polymer (A) contains 0.25 to 5 mmol/g of amine oxide group.

[11] A biofilm formation inhibiting laminate comprising a coating film comprising the biofilm formation inhibiting coating agent according to [9] or [10] on a base material.

[12] A cell culture equipment treatment agent comprising the protein, cell or microorganism adhesion inhibitor according to any one of [1] to [4].

[13] The cell culture equipment treatment agent according to [12], wherein the vinyl polymer (A) contains 0.25 to 5 mmol/g of amine oxide group.

[14] The cell culture equipment treatment agent according to [12] or [13], wherein the vinyl polymer (A) has at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group.

[15] The cell culture equipment treatment agent according to [14], further comprising a crosslinking agent.

[16] A cell culture device having, on a substrate, a cell adhesion prevention film containing the protein, cell or microorganism adhesion inhibitor according to any one of [1] to [4].

[17] A medical device comprising the cell culture equipment according to [16].

[18] A protein stabilizer comprising the protein, cell or microorganism adhesion inhibitor according to any one of [1] to [4].

[19] The protein stabilizer according to [18], wherein the vinyl polymer (A) contains 1 to 10 mmol/g of amine oxide group.

[20] A method for stabilizing a protein, which comprises coexisting the protein stabilizer according to [18] or [19] with the protein.

[21] A cell culture medium additive comprising the protein, cell or microorganism adhesion inhibitor according to any one of [1] to [4].

[22] The cell culture medium additive according to [21], wherein the vinyl polymer (A) contains 1 to 10 mmol/g of amine oxide group.

[23] The cell culture medium additive according to [21] or [22], wherein the cells are antibody-producing cells.

[24] A cell culture medium comprising the cell culture medium additive according to any one of [21] to [23].

[25] The cell culture medium according to [24], wherein the concentration of the vinyl polymer (A) in the cell culture medium is 0.01% by mass or more and 1% by mass or less.

[26] A method for producing a cell aggregate, comprising culturing cells in a cell culture medium containing the cell culture medium additive according to any one of [21] to [23] to form a cell aggregate.

According to the present invention, it is possible to provide an adhesion inhibitor for proteins, cells, or microorganisms that exhibits an excellent ability to inhibit adhesion of proteins, cells, or microorganisms, and various uses containing the adhesion inhibitor.

In addition, the blocking agent of the present invention has the ability not to interfere with color development due to specific adsorption of the stain to the target protein, sufficiently covers proteins other than the target, and prevents non-specific adsorption of the stain to proteins other than the target. Unlike blocking agents derived from living organisms, it can stably exhibit the ability to prevent color development. By using this blocking agent, it becomes possible to perform sensitive biochemical analyzes using antigen-antibody reactions such as Western blotting and immunohistological staining.

In addition, the present invention has the effect of sufficiently covering proteins other than the target without hindering color development due to specific adsorption of the stain to the target protein, preventing non-specific adsorption of the stain to proteins other than the target, and preventing color development. It is possible to provide a blocking agent for biochemical analysis that exhibits the following properties. This makes it possible to perform sensitive biochemical analyzes using antigen-antibody reactions such as Western blotting, immunohistological staining, ELISA, and immunochromatography. For example, in immunohistological staining, a blocking agent for biochemical analysis of the present invention is brought into contact with a pathological tissue fixed on the surface of a base material, and then a staining antibody is adsorbed to the antigen in the pathological tissue to detect the antigen. can.

Further, according to the present invention, it is possible to provide a biofilm formation inhibiting coating agent that is excellent in safety, coatability, and water resistance, and enables long-term inhibition of biofilm formation.
The biofilm formation-inhibiting coating agent of the present invention can be suitably used on the surfaces of substances to which microorganisms are expected to adhere and biofilms to form, such as medical equipment, manufacturing equipment, or the inner surface of water tanks.

Further, according to the present invention, it is possible to provide a cell culture equipment treatment agent that has low cytotoxicity and has an excellent effect of preventing adhesion of proteins and cells, and to provide a cell culture equipment that can easily produce cell aggregates. , a method for manufacturing a medical device and cell culture equipment can be provided.

In addition, by using a vinyl polymer (A) that has an amine oxide group and a mass average molecular weight of 2,000 or more, we have created a protein stabilizer that can be stored stably without impairing protein functions. can be provided. Furthermore, by allowing the protein stabilizer and the protein to coexist, it is possible to provide a method for stabilizing the protein, which allows stable storage without impairing the function of the protein.

In addition, by adding a vinyl polymer (A) that has an amine oxide group and a mass average molecular weight of 2,000 or more to the culture medium as a cell culture medium additive, it can be used without increasing cost or labor. It becomes possible to easily produce cell aggregates. That is, the present invention provides a cell culture medium additive for producing a three-dimensional cell aggregate with a good proportion of viable cells without increasing cost or labor, and a cell culture medium containing the additive. , a method for producing cell aggregates using the additive can be provided.

In addition, adding a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 2,000 or more to the culture medium as an additive to the antibody-producing cell culture medium increases cost and labor. Antibodies can be efficiently produced without Furthermore, the generation of antibody-producing cell aggregates during culture is suppressed, making it easier to purify the desired antibody. That is, the present invention provides an antibody-producing cell culture medium additive, a method for culturing antibody-producing cells using the additive, and a cell culture medium additive for increasing antibody productivity and purification without increasing cost or labor. Culture media and methods for producing antibodies can be provided.

The adhesion inhibitor for proteins, cells, or microorganisms of the present invention is characterized by containing a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 2,000 or more. By having the vinyl polymer (A), it exhibits excellent adhesion inhibiting properties for proteins, cells, or microorganisms.

<Vinyl polymer (A)>
The vinyl polymer (A) of the present invention only needs to have an amine oxide group and a mass average molecular weight of 2,000 or more, and conventionally known polymers can be used, or two or more types can be used in combination. Good too. An image of the amine oxide group is shown in General Formula 4 below. In the formula, R ¹⁰ , R ¹¹ and R ¹² each independently represent an organic group.

In the vinyl polymer (A) having an amine oxide group, at least one of the R ¹⁰ , R ¹¹ , and R ¹² is linked to the polymer.

The method of introducing the amine oxide group into the vinyl polymer (A) is not particularly limited, but for example,
(1) Polymerize a monomer having an ethylenically unsaturated group containing as an essential component a monomer (a) having one ethylenically unsaturated group and a tertiary amino group in one molecule, and oxidize the obtained polymer. A method of oxidizing with an agent,
(2) A method of polymerizing a monomer having an ethylenically unsaturated group containing as an essential component the monomer (b) having one ethylenically unsaturated group and an amine oxide group in one molecule.

In addition, as a method other than the above, for example, an unsaturated compound containing an epoxy group such as glycidyl (meth)acrylate or an unsaturated compound containing an isocyanate group such as 2-isocyanate ethyl (meth)acrylate, and hydroxyethyl-N,N- Copolymerization using a reaction product with an amine oxide group-containing compound such as dimethylamine oxide can also be used.

For example, when the amine oxide group is introduced by method (1), the content of the amine oxide group can be calculated using the following formula 1.

Ｂ：アミンオキシド変換率は、酸化反応前に滴定により求めたアミン価がどの程度減少したかを計算により求めることで算出することができる。

B: The amine oxide conversion rate can be calculated by calculating how much the amine value determined by titration has decreased before the oxidation reaction.

For example, when the amine oxide group is introduced by method (2), the content of the amine oxide group can be determined from the amount of the monomer having the amine oxide group used in the polymerization.

In the present invention, the amine oxide group preferably corresponds to one of the following general formulas 1 to 3.

[In general formulas 1 to 3,
X is a divalent bonding group,
y is 0 or 1,
R ¹ is an alkylene group having 1 to 6 carbon atoms,
R ² and R ³ are each independently an alkyl group having 1 to 4 carbon atoms,
R ⁴ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Four of R ⁵ to R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ⁵ to R ⁹ is a bond with the main chain of the vinyl polymer (A). represents the bond position,
* represents the bonding position with the main chain of the vinyl polymer (A). ]

<Monomer (a)>
Monomer (a) is a monomer having one ethylenically unsaturated group and a tertiary amino group having an unpaired electron pair and not being substituted with a hydrogen atom in one molecule. A tertiary amino group can be converted into an amine oxide group by being oxidized with various oxidizing agents.

The monomer (a) is not particularly limited, but examples of monomers for forming the structure of general formula 1 include N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N,N-dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N- Dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, vinyl N,N-dimethylaminopropionate, vinyl N,N-diethylaminopropionate, N,N-dimethylacrylamide, N,N-dimethyl Allylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylaminoethylstyrene, N,N-dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinyl amine,
Alternatively, a reaction product of an unsaturated group-containing acid anhydride such as maleic anhydride, itaconic anhydride, or citraconic anhydride with N,N-dimethyl-1,3-propanediamine, etc.
Examples include reaction products of epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propanediamine.
In the present invention, "(meth)acrylic" refers to both "acrylic" and "methacrylic".

Examples of compounds for forming the structure of general formula 3 include 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-vinylpyridine, -Methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2- Examples include (t-butyl)-5-vinylpyridine.

Examples of compounds for forming the structure of general formula 2 include 1-vinylimidazole,
Examples include 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4-(t-butyl)-1-vinylimidazole, and the like.

The tertiary amine group derived from monomer (a) can be converted into an amine oxide group by using various oxidizing agents. Thereby, an amine oxide group can be introduced into the vinyl polymer (A).

Examples of the oxidizing agent include, but are not limited to, peroxides such as hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, and t-butyl hydroperoxide, and ozone. .

<Monomer (b)>
Monomer (b) refers to a monomer having one ethylenically unsaturated group and one amine oxide group in one molecule. By polymerizing the monomer (b) having an amine oxide group, the amine oxide group can be incorporated into the vinyl polymer (A). The monomer (b) is not particularly limited, but examples include those obtained by substituting the tertiary amine group of the monomer (a) with an amine oxide group.

<Monomer (c)>
Monomer (c) is a monomer having one ethylenically unsaturated group and an alkyl group having 4 to 18 carbon atoms in one molecule. Since the vinyl polymer (A) has an alkyl group having 4 to 18 carbon atoms, the polarity can be controlled and the adsorption performance to proteins other than the target can be improved.

Monomer (c) is not particularly limited, but includes butyl (meth)acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, and nonyl (meth)acrylate. , alkyl (meth)acrylates such as decyl (meth)acrylate, dodecyl (meth)acrylate, cyclohexyl (meth)acrylate;
Examples include α-olefin ethylenically unsaturated monomers such as 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.

<Monomer (d)>
In the present invention, monomer (d) has one ethylenically unsaturated group in one molecule other than monomers (a), (b), and (c) that are copolymerizable with monomers (a), (b, and c). Indicates a monomer with

The monomer (d) is not particularly limited, but
Alkyl (meth)acrylates such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, stearyl (meth)acrylate, cetyl (meth)acrylate, isobornyl (meth)acrylate, methoxyethyl (meth)acrylate, ethoxy Acrylic ester (meth)acrylates such as ethyl (meth)acrylate, propoxyethyl (meth)acrylate, butoxyethyl (meth)acrylate, ethoxypropyl (meth)acrylate;
Aromatic ester (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, phenoxyethyl (meth)acrylate;
Allyl (meth)acrylate, 1-methylallyl (meth)acrylate, 2-methylallyl (meth)acrylate, 1-butenyl (meth)acrylate, 2-butenyl (meth)acrylate, 3-(meth)acrylate Butenyl, 1,3-methyl-3-butenyl (meth)acrylate, 2-chlorallyl (meth)acrylate, 3-chlorallyl (meth)acrylate, o-allylphenyl (meth)acrylate, (meth)acrylic acid 2-(allyloxy)ethyl, allyllactyl (meth)acrylate, citronellyl (meth)acrylate, geranyl (meth)acrylate, rhodinyl (meth)acrylate, cinnamyl (meth)acrylate, diallyl maleate, diallylitaconic acid, Ethylenically unsaturated group-containing (meth)acrylic acid esters such as vinyl (meth)acrylate, vinyl crotonate, vinyl oleate, vinyl linolenate, and 2-(2'-vinyloxyethoxy)ethyl (meth)acrylate. ;
Perfluoromethylmethyl (meth)acrylate, perfluoroethylmethyl (meth)acrylate, 2-perfluorobutylethyl (meth)acrylate, 2-perfluorohexylethyl (meth)acrylate, 2-perfluorooctylethyl (meth)acrylate , 2-perfluoroisononylethyl (meth)acrylate, 2-perfluorononylethyl (meth)acrylate, 2-perfluorodecylethyl (meth)acrylate, perfluoropropylpropyl (meth)acrylate, perfluorooctylpropyl (meth)acrylate ) acrylate, perfluorooctyl amyl (meth)acrylate, perfluorooctyl undecyl (meth)acrylate, etc. (meth) such as perfluoroalkyl group-containing ethylenically unsaturated monomers having a perfluoroalkyl group having 1 to 20 carbon atoms. Acrylate monomer;
2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-(meth)acryloyloxyethyl-2-hydroxyethylphthalate, glycerol mono(meth)acrylate , 4-hydroxyvinylbenzene, 1-ethynyl-1-cyclohexanol, allyl alcohol, and other hydroxyl group-containing monomers;
Maleic acid, fumaric acid, itaconic acid, citraconic acid, or alkyl or alkenyl monoesters thereof, β-(meth)acryloxyethyl phthalate monoester, β-(meth)acryloxyethyl isophthalate monoester, terephthalic acid A monomer having a carboxylic acid group, such as β-(meth)acryloxyethyl monoester, β-(meth)acryloxyethyl succinate monoester, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, or its anhydride;
Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , monomers having primary to tertiary amide groups such as diacetone (meth)acrylamide;
(meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamido-1,1-dimethylpropyl) ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl) ammonium chloride, and a monomer having a quaternary amino group such as trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride;
Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate Acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth)acrylate , n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexaethylene Monomers having polyether chains such as glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate;
(Meth)acrylate monomers with a polymer structure in the side chain, such as ethylenically unsaturated compounds with polyester chains such as lactone-modified (meth)acrylate; styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allyl Aromatic vinyl monomers such as benzene and ethynylbenzene; ethylenically unsaturated monomers containing nitrile groups such as (meth)acrylonitrile; vinyl acetate, vinyl butyrate, vinyl propionate, vinyl hexanoate, vinyl caprylate, vinyl laurate, palmitic acid Fatty acid vinyl compounds such as vinyl and vinyl stearate; vinyl ether type ethylenically unsaturated monomers such as butyl vinyl ether and ethyl vinyl ether; allyl monomers such as allyl acetate and allyl cyanide; vinyl cyanide, vinyl cyclohexane, vinyl methyl ketone, etc. Vinyl monomers; ethynyl monomers such as acetylene and ethynyltoluene; perfluoroalkyls such as perfluorobutylethylene, perfluorohexylethylene, perfluorooctylethylene, and perfluorodecylethylene; perfluoroalkyl group-containing ethylenically unsaturated compounds such as alkylenes; Examples include monomers having ethylenically unsaturated bonds that are not (meth)acrylates, such as.

Below, various uses including adhesion inhibitors for proteins, cells, or microorganisms are detailed.

<Blocking agent for biochemical analysis>
The blocking agent of the present invention contains the above-mentioned vinyl polymer (A).
In the present invention, the content of amine oxide groups is preferably 1 to 10 mmol/g, more preferably 2 to 8 mmol/g. By having the content of amine oxide groups within the above range, it is possible to exhibit suitable water solubility, suppress adsorption to proteins that are the target of staining, and improve adsorption performance to proteins other than the target.

In the present invention, the amine oxide group preferably corresponds to one of the above-mentioned general formulas 1 to 3, and by having the above structure, it exhibits suitable water solubility and has good adsorption to the protein that is the target of staining. It is possible to improve the adsorption performance for proteins other than the target while suppressing the adsorption performance.

In the present invention, when an amine oxide group is introduced into the vinyl polymer (A) by method (1), it is desirable to obtain it by polymerizing monomers containing 20 to 99.9% by weight of monomer (a) based on the total monomers.

In the present invention, when an amine oxide group is introduced into the vinyl polymer (A) by method (2), it is desirable to obtain the monomer by polymerizing a monomer containing 20 to 99.9% by weight of the monomer (b) based on the total monomers.

In the present invention, when an amine oxide group is introduced into the vinyl polymer (A) by method (1), it is desirable to obtain the monomer by polymerizing a monomer containing 0.1 to 80% by weight of the monomer (c) based on the total monomers. In the present invention, when an amine oxide group is introduced into the vinyl polymer (A) by method (2), it is desirable to obtain the monomer by polymerizing a monomer containing 0.1 to 80% by weight of the monomer (c) based on the total monomers.

<Copolymer consisting of thiol group-containing compound (x) and vinyl monomer>
Furthermore, the blocking agent for biochemical analysis of the present invention contains a thiol group-containing compound (x) and vinyl
It may contain an amine oxide group-containing vinyl polymer (A) (hereinafter also referred to as a vinyl polymer), which is a copolymer consisting of a vinyl monomer and a vinyl monomer.
By having an amine oxide group, it is possible to exhibit suitable water solubility and improve adsorption performance to proteins other than the target while suppressing adsorption to the protein that is the target of staining. Further, by using the thiol group-containing compound (x), a sulfur atom derived from a thiol group is introduced into the copolymer, and a branched structure is further introduced by using a trifunctional or higher-functional thiol group-containing compound described below. The introduction of sulfur atoms and branched structures can approximate the structure of proteins of biological origin, such as bovine serum albumin, or the state dissolved in an aqueous solution.
As described above, the blocking agent for biochemical analysis of the present invention containing the amine oxide group-containing vinyl polymer (A), which is a copolymer consisting of the thiol group-containing compound (x) and a vinyl monomer, properties are controlled and exhibits excellent blocking performance.

Among these, the amine oxide group-containing vinyl polymer, which is a copolymer consisting of the thiol group-containing compound (x) and a vinyl monomer, is preferably one of the following (i) or (i) either.
(i) A copolymer consisting of a vinyl monomer containing an amine oxide group-containing vinyl monomer (a1) and a thiol group-containing compound (x).
(ii) reacting an oxidizing agent with a tertiary amino group of a copolymer consisting of a vinyl monomer containing a vinyl monomer (a2) having a tertiary amino group and a thiol group-containing compound (x); copolymer.
That is, the method for producing the amine oxide group-containing vinyl polymer (A) is not particularly limited, but from the viewpoint of producing and purifying the polymer, preferably it is either (1) or (2) below.
(1) A method of copolymerizing a vinyl monomer containing an amine oxide group-containing vinyl monomer (a1) and a thiol group-containing compound (x).
(2) An oxidizing agent is added to the tertiary amino group of a copolymer obtained by copolymerizing a vinyl monomer containing a vinyl monomer (a2) having a tertiary amino group and a thiol group-containing compound (x). How to react.
Furthermore, the copolymer (ii) is preferred from the viewpoint of easier production and purification of the polymer, and (2) is preferred as the production method.

<Vinyl monomer>
The amine oxide group-containing vinyl polymer, which is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, which will be described later. , an amine oxide group can be introduced using a vinyl monomer. Specifically, an amine oxide group may be introduced using an amine oxide group-containing vinyl monomer (a1), or after obtaining a copolymer using a vinyl monomer (a2) having a tertiary amino group, The tertiary amino group may be modified into an amine oxide group by reacting with an oxidizing agent.
The vinyl monomer is preferably a (meth)acrylate group or an aromatic vinyl group from the viewpoint of polymerizability.
Each vinyl monomer will be explained below.

[Amine oxide group-containing vinyl monomer (a1)]
The amine oxide group-containing vinyl monomer (a1) is not particularly limited, and a commercially available product or a synthetic product may be used. ) may be reacted with an oxidizing agent described below to form an amine oxide group.

In the present invention, when an amine oxide group is introduced by method (1) into an amine oxide group-containing vinyl polymer, which is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, the amine oxide group-containing vinyl It is desirable that the monomer (a1) be contained in an amount of 20 to 99.9% by weight based on the total vinyl monomers.

[Vinyl monomer (a2) having tertiary amino group]
The vinyl monomer (a2) having a tertiary amino group can have an amine oxide structure by reacting the tertiary amino group with an oxidizing agent described below.

In the present invention, when an amine oxide group is introduced by method (2) into an amine oxide group-containing vinyl polymer, which is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, a tertiary amino group is introduced. It is desirable that the vinyl monomer (a2) is contained in an amount of 20 to 99.9% by weight based on the total vinyl monomers.

The vinyl monomer (a2) having a tertiary amino group is not particularly limited, but examples of the vinyl monomer (a2) for forming the structure of general formula 1 include:
N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N,N- Dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylaminopropionic acid Vinyl, N,N-diethylaminopropionate vinyl, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylaminoethylstyrene, N,N-dimethylvinyl Amine, N,N-diethylvinylamine, N,N-diphenylvinylamine,
Alternatively, a reaction product of an unsaturated group-containing acid anhydride such as maleic anhydride, itaconic anhydride, or citraconic anhydride with N,N-dimethyl-1,3-propanediamine, etc.
Examples include reaction products of epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propanediamine.

For example, to form the structure of general formula 3,
2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine , 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2-(t-butyl)-5-vinylpyridine, and the like.

For example, to form the structure of general formula 2,
Examples include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4-(t-butyl)-1-vinylimidazole, and the like.

[Other vinyl polymers]
The vinyl monomer (a) may contain other vinyl monomers other than the amine oxide group-containing vinyl monomer (a1) and the tertiary amino group-containing vinyl monomer (a2).

(Vinyl monomer (a3) having an alkyl group having 1 to 18 carbon atoms)
As other vinyl monomers, it is preferable to use a vinyl monomer (a3) having an alkyl group having 1 to 18 carbon atoms. By introducing an alkyl group having 1 to 18 carbon atoms into the vinyl polymer (A), the polarity can be controlled and the adsorption performance to proteins other than the target can be improved.

The vinyl monomer (a3) having an alkyl group having 1 to 18 carbon atoms is not particularly limited, but includes, for example, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, etc. ) acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate, cyclohexyl (meth)acrylate, ) acrylate, alkyl (meth)acrylates such as isobornyl (meth)acrylate, stearyl (meth)acrylate, cetyl (meth)acrylate;
Examples include α-olefinic ethylenically unsaturated monomers such as 1-propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.

In the present invention, the vinyl monomer (a3) having an alkyl group having 1 to 18 carbon atoms is preferably contained in an amount of 0.1 to 80% by weight based on the total vinyl monomer.

Vinyl monomers that may be used in addition to the above-mentioned vinyl monomers (a1) to (a3) are not particularly limited, but include, for example,
Alkoxyalkyl (meth)acrylates such as methoxyethyl (meth)acrylate, ethoxyethyl (meth)acrylate, propoxyethyl (meth)acrylate, butoxyethyl (meth)acrylate, ethoxypropyl (meth)acrylate;
Aromatic-containing (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, and phenoxyethyl (meth)acrylate;
Allyl (meth)acrylate, 1-methylallyl (meth)acrylate, 2-methylallyl (meth)acrylate, 1-butenyl (meth)acrylate, 2-butenyl (meth)acrylate, 3-(meth)acrylate Butenyl, 1,3-methyl-3-butenyl (meth)acrylate, 2-chlorallyl (meth)acrylate, 3-chlorallyl (meth)acrylate, o-allylphenyl (meth)acrylate, (meth)acrylic acid 2-(allyloxy)ethyl, allyllactyl (meth)acrylate, citronellyl (meth)acrylate, geranyl (meth)acrylate, rhodinyl (meth)acrylate, cinnamyl (meth)acrylate, diallyl maleate, diallylitaconic acid, Ethylenically unsaturated group-containing (meth)acrylic acid esters such as vinyl (meth)acrylate, vinyl crotonate, vinyl oleate, vinyl linolenate, and 2-(2'-vinyloxyethoxy)ethyl (meth)acrylate. ;
Perfluoromethylmethyl (meth)acrylate, perfluoroethylmethyl (meth)acrylate, 2-perfluorobutylethyl (meth)acrylate, 2-perfluorohexylethyl (meth)acrylate, 2-perfluorooctylethyl (meth)acrylate , 2-perfluoroisononylethyl (meth)acrylate, 2-perfluorononylethyl (meth)acrylate, 2-perfluorodecylethyl (meth)acrylate, perfluoropropylpropyl (meth)acrylate, perfluorooctylpropyl (meth)acrylate ) acrylate, perfluorooctyl amyl (meth)acrylate, perfluorooctyl undecyl (meth)acrylate, etc. (meth) such as perfluoroalkyl group-containing ethylenically unsaturated monomers having a perfluoroalkyl group having 1 to 20 carbon atoms. Acrylate monomer;
2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-(meth)acryloyloxyethyl-2-hydroxyethylphthalate, glycerol mono(meth)acrylate , 4-hydroxyvinylbenzene, 1-ethynyl-1-cyclohexanol, allyl alcohol, and other hydroxyl group-containing monomers;
Maleic acid, fumaric acid, itaconic acid, citraconic acid, or alkyl or alkenyl monoesters thereof, β-(meth)acryloxyethyl phthalate monoester, β-(meth)acryloxyethyl isophthalate monoester, terephthalic acid A monomer having a carboxylic acid group, such as β-(meth)acryloxyethyl monoester, β-(meth)acryloxyethyl succinate monoester, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, or its anhydride;
Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , monomers having primary to tertiary amide groups such as diacetone (meth)acrylamide;
(meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamido-1,1-dimethylpropyl) ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl) ammonium chloride, and a monomer having a quaternary amino group such as trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride;
Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate Acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth)acrylate , n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexaethylene Monomers having polyether chains such as glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate;
(meth)acrylate monomers having a polymer structure in their side chains, such as ethylenically unsaturated compounds having polyester chains such as lactone-modified (meth)acrylates;
Aromatic vinyl monomers such as styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allylbenzene, ethynylbenzene;
Ethylenically unsaturated monomers containing nitrile groups such as (meth)acrylonitrile;
Fatty acid vinyl compounds such as vinyl acetate, vinyl butyrate, vinyl propionate, vinyl hexanoate, vinyl caprylate, vinyl laurate, vinyl palmitate, vinyl stearate; vinyl ether type ethylenically unsaturated monomers such as butyl vinyl ether, ethyl vinyl ether, etc. ;
Allyl monomers such as allyl acetate and allyl cyanide;
Vinyl monomers such as vinyl cyanide, vinylcyclohexane, and vinyl methyl ketone; Ethynyl monomers such as acetylene and ethynyltoluene; perfluoroalkyl and alkylenes such as perfluorobutylethylene, perfluorohexylethylene, perfluorooctylethylene, and perfluorodecylethylene; Examples include monomers having ethylenically unsaturated bonds that are not (meth)acrylates, such as perfluoroalkyl group-containing ethylenically unsaturated compounds.

The amine oxide group-containing vinyl polymer, which is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, is a copolymer consisting of an amine oxide group-containing vinyl monomer (a1) or a vinyl monomer having a tertiary amino group ( 20 to 99.9% by weight of a2), 0.1 to 80% by weight of a vinyl monomer (a3) having an alkyl group having 1 to 18 carbon atoms, and other ethylenically unsaturated group monomers (c). It is preferably contained in a range of 0 to 79.9% by weight.

<Oxidizing agent>
The oxidizing agent is not particularly limited, but oxidizing agents such as peroxide or ozone are preferably used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, t-butyl hydroperoxide, etc. Hydrogen peroxide is preferred, and is usually used in an aqueous solution. used in the form Since peroxide also has a function as a radical generator, in the case of a vinyl copolymer obtained by copolymerizing a vinyl monomer containing a tertiary amino group, it is preferable to oxidize the peroxide after polymerization.

<Amine oxide group content>
The amine oxide group imparts water solubility and non-protein adsorption properties to the copolymer. The amine oxide group content in the amine oxide group-containing vinyl polymer, which is a copolymer consisting of the thiol group-containing compound (x) and a vinyl monomer, is preferably 1 to 10 mmol/g, more preferably 2 ~8 mmol/g. By being within the above range, it is possible to exhibit suitable water solubility and improve adsorption performance to proteins other than the target while suppressing adsorption to the protein that is the target of staining.
The content of amine oxide groups is the content of at least one of the amine oxide structures represented by the above general formulas 1 to 3, and when a copolymer is obtained by polymerizing the amine oxide group-containing vinyl monomer (a1). can be determined from the amount of the amine oxide group-containing vinyl monomer (a1) used in the polymerization. On the other hand, when the polymer obtained after polymerizing the tertiary amino group-containing vinyl monomer (a2) is oxidized, it can be calculated using the above-mentioned formula 1.

<Production of an amine oxide group-containing vinyl polymer that is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer>
The amine oxide group-containing vinyl copolymer, which is a copolymer consisting of a thiol group-containing compound (x) and a vinyl monomer, is produced by copolymerizing a vinyl monomer using a thiol group-containing compound (x) as a chain transfer agent. It can be manufactured by By using the thiol group-containing compound (x), sulfur atoms can be incorporated into the amine oxide group-containing vinyl polymer, and by using the trifunctional or higher functional thiol group-containing compound, branching can be achieved in the structure of the polymer. structure can be introduced. By adopting such a structure, it is possible to approximate the dissolved state of a biologically derived protein in an aqueous solution, so it is presumed that excellent blocking performance can be obtained.

<Thiol group-containing compound (x)>
The thiol group-containing compound (x) may be any compound containing a thiol that acts as a chain transfer agent in the polymerization reaction. Among these, those having a plurality of thiol groups are particularly preferred. Examples of thiol group-containing compounds having multiple thiol groups include:
2-Mercaptoethanol, β-mercaptopropionic acid, 2-ethylhexyl-3-mercaptopropionate, n-octyl-3-mercaptopropionate, methoxybutyl-3-mercaptopropionate, stearyl-3-mercaptopropionate ate, thioglycerol, trimethylolpropane tris(3-mercaptopropionate), tris-[(3-mercaptopropionyloxy)-ethyl]-isocyanurate, pentaerythritol tetrakis(3-mercaptopropionate), tetraethylene glycol Bis(3-mercaptopropionate), dipentaerythritol hexakis(3-mercaptopropionate), and the like. Among these, compounds containing trifunctional or higher functional thiol groups are preferred for the reasons mentioned above.

[Trifunctional or higher thiol group-containing chain transfer agent (x)]
The compound containing a trifunctional or higher functional thiol group may have a trifunctional or higher functional thiol group (-SH group) that acts as a chain transfer agent in the molecule, and examples of such compounds include the compound represented by the following general formula 5. Can be mentioned.

General formula 5

[In General Formula 5, R ⁵ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, or an isocyanurate group. n each independently represents an integer from 0 to 10, and k ¹ represents an integer from 0 to 10.
3 represents an integer, l ¹ represents an integer from 0 to 4, m ¹ represents an integer from 0 to 3, k ² represents an integer from 0 to 3, l ² represents an integer from 0 to 3, m ² represents an integer from 0 to 3. However, the sum of k ¹ , l ¹ and m ¹ is 4 or less, the sum of k ² , l ² and m ² is 4 or less, and the sum of l ¹ and l ² is 3 or more. ]

Examples of the alkyl group represented by R ⁵ in general formula 5 include alkyl groups having 1 to 10 carbon atoms, such as methyl group, ethyl group, n-propyl group, i-propyl group, t-butyl group, etc. can be mentioned. Examples of the alkoxy group represented by R ⁵ in general formula 5 include alkoxy groups having 1 to 10 carbon atoms, such as methoxy group, ethoxy group, i-propoxy group, t-butoxy group, and n-octyloxy group. , 2-methoxyethoxy group and the like. The aryl group represented by R ⁵ in General Formula 5 includes aryl groups having 6 to 30 carbon atoms, such as phenyl and naphthyl groups.

Specific examples of trifunctional or more functional thiol group-containing compounds are listed below, but the thiol-containing compounds that can be employed in the present invention are not limited to these specific examples.

The weight average molecular weight (Mw) of the amine oxide group-containing vinyl polymer is determined by the amount of the thiol group-containing compound (x) added during the polymerization reaction. Therefore, the amount of the thiol group-containing compound (x) added is preferably 0.1% to 15% by weight, more preferably 0.1% to 15% by weight, based on the total vinyl monomers constituting the amine oxide group-containing vinyl polymer. .1% to 10% by weight. When the content is 0.1% by weight or more, heat control and molecular weight control during the polymerization reaction can be easily controlled, and when the content is 15% by weight or less, better blocking performance is exhibited.

<Weight average molecular weight (Mw) of amine oxide group-containing vinyl polymer (A)>
The weight average molecular weight (Mw) of the amine oxide group-containing vinyl polymer (A) is preferably 1,000 to 10,000,000. When the weight average molecular weight (Mw) is 1,000 or more, non-specific adsorption of substances other than the target can be suppressed, and when it is 10,000,000 or less, it can be made water-soluble.

The weight average molecular weight of the amine oxide group-containing vinyl polymer is determined by gel permeation chromatography (GPC) in terms of standard polystyrene. The measurement device and measurement conditions were basically based on Condition 1 below. However, depending on the type of polymer, a suitable carrier (eluent) and a column suitable for the carrier may be selected and used. For other matters, please refer to JISK7252-1~4:2008. In addition, if it is difficult to measure the molecular weight of the amine oxide group-containing vinyl polymer, the weight average of the amine oxide group-containing vinyl polymer and the tertiary amino group-containing vinyl polymer that is the precursor before the reaction with the amine oxidizing agent. The molecular weight can be the weight average molecular weight of the amine oxide group-containing vinyl polymer. The weight average molecular weight of the vinyl polymer having a tertiary amino group, which is a precursor of the amine oxide group-containing vinyl polymer before the reaction with the amine oxidizing agent, was measured in terms of standard polystyrene by gel permeation chromatography (GPC). The measurement device and measurement conditions were basically based on Condition 2 below.
(Condition 1)
Column: ShodexOHpark SB-800RL
ShodexOHpark SB-800RH
Shodex0Hpark SB-802.5 HQ
Shodex0Hpark SB-806M HQ
Carrier using a column connected to: Phosphate buffer aqueous solution Measurement temperature: 40°C
Sample concentration: 0.2% by mass
Detector: RI (refractive index) detector (condition 2)
Column: TOSOHTSKgelSuperAW4000,
TOSOHTSKgelSuperAW3000,
TOSOHTSKgelSuperAW2500
Use a column that connects
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Sample concentration: 0.2% by mass
Detector: RI (refractive index) detector

<Biofilm formation inhibiting coating agent>
The vinyl polymer contained in the biofilm formation inhibiting coating agent preferably contains an amine oxide group and has a mass average molecular weight of 2,000 to 10,000,000. By having an amine oxide group, it exhibits an excellent effect of inhibiting biofilm formation.

In the present invention, the vinyl polymer having amine oxide groups can be obtained by the following two methods. That is, a monomer having an amine oxide group and another monomer can be polymerized to obtain a polymer having an amine oxide group. Alternatively, after obtaining a polymer having a tertiary amino group, which can be called a precursor functional group for an amine oxide group, the tertiary amino group can be reacted with an oxidizing agent to introduce an amine oxide group into the polymer. The latter method is preferred because it is less likely to cause a reaction. Note that reacting a tertiary amino group with an oxidizing agent is also referred to as "oxidation" hereinafter.

Specifically, the vinyl polymer preferably contains at least one of the structures represented by the above-mentioned general formulas 1 to 3, and among them, those containing the structure represented by general formula 1 are particularly preferable.

Vinyl polymers having such a structure can be obtained in two ways, as described above. More preferably, the tertiary amino group-containing monomer (A) is oxidized and then polymerized with another monomer, or the tertiary amino group-containing monomer (A) and another monomer are polymerized and then oxidized. It is.

<Tertiary amino group-containing monomer (A)>
Among the tertiary amino group-containing monomers (A) as precursors before oxidation, those for forming the structure of general formula 1 include:
For example, N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N, N-dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylamino Vinyl propionate, N,N-diethylaminovinyl propionate, N,N-dimethylacrylamide, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylamino Ethylstyrene, N,N-dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinylamine,
Alternatively, a reaction product of an unsaturated group-containing acid anhydride such as maleic anhydride, itaconic anhydride, or citraconic anhydride with N,N-dimethyl-1,3-propanediamine, etc.
Examples include reaction products of epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propanediamine.
Preferably, it is a (meth)acrylate monomer, more preferably N,N-dialkylaminoethyl (meth)acrylate or N,N-dialkylaminopropyl (meth)acrylate.
In the present invention, "(meth)acrylic" refers to both "acrylic" and "methacrylic."

Examples of compounds for forming the structure of general formula 3 include 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-vinylpyridine, -Methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2- Examples include (t-butyl)-5-vinylpyridine.

Examples of substances for forming the structure of general formula 2 include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4- Examples include (t-butyl)-1-vinylimidazole.

<Monomer (B) having a crosslinkable group>
From the viewpoint of improving the water resistance of the vinyl polymer, when obtaining the vinyl polymer, in addition to the monomer (A), a carboxyl group, a hydroxyl group, an epoxy group, a primary or secondary amino group, and an isocyanate group are selected. It is preferable to use a monomer (B) having at least one crosslinkable group. The crosslinkable group derived from the monomer (B) reacts with the crosslinking agent described below to introduce a crosslinked structure into the polymer coating film, thereby exhibiting excellent water resistance.

For example, a copolymer into which a carboxyl group has been introduced can be crosslinked with an epoxy compound, an aziridine compound, a carbodiimide compound, or an N-hydroxyethylacrylamide compound. The copolymer into which hydroxyl groups have been introduced can be crosslinked with an isocyanate compound or the like. A copolymer into which amino groups have been introduced can be crosslinked with an epoxy compound. The copolymer into which isocyanate groups have been introduced can be crosslinked with a hydroxyl group-containing compound.

The carboxyl group-containing monomer is not particularly limited as long as it has a carboxyl group in its structure; for example, (meth)acrylic acid, 2-carboxyethyl (meth)acrylate, or alkylene such as ethylene oxide or propylene oxide. Examples include alkylene oxide-added succinic acid (meth)acrylate having a carboxyl group at the end of which oxide is repeatedly added.

The hydroxyl group-containing monomer is not particularly limited as long as it has a hydroxyl group in its structure, and examples include 2-hydroxyethyl (meth)acrylate, 1-hydroxypropyl (meth)acrylate, and (meth)acrylic acid. 2-Hydroxypropyl, 3-hydroxypropyl (meth)acrylate, 1-hydroxybutyl (meth)acrylate, monofunctional glycerol (meth)acrylate, polylactone type (meth) having a hydroxyl group at the end by ring-opening addition of a lactone ring. ) Acrylic esters, alkylene oxide-added (meth)acrylic esters having a hydroxyl group at the end obtained by repeatedly adding alkylene oxides such as ethylene oxide or propylene oxide, and glucose ring-based (meth)acrylic esters.

The epoxy group-containing monomer is not particularly limited as long as it has an epoxy group in its structure, and examples thereof include glycidyl (meth)acrylate, 3,4-epoxycyclohexylmethyl (meth)acrylate, etc. .

The monomer containing a primary or secondary amino group is not particularly limited as long as it has an amino group in its structure; for example, monomethylaminoethyl (meth)acrylate, monoethylaminoethyl (meth)acrylate. , monomethylaminopropyl (meth)acrylate, monoethylaminopropyl (meth)acrylate, and the like.

The monomer (B) having a crosslinking group is preferably a carboxyl group-containing monomer from the viewpoint of water resistance and long-term biofilm formation inhibiting ability.

<Other monomers (C)>
When obtaining a vinyl polymer, other monomers (C) having one ethylenically unsaturated group in one molecule can be used in addition to the monomers (A) and (B). By introducing a structure based on the monomer (C), polarity and Tg can be appropriately controlled, and it is possible to have excellent coating properties, water resistance, and weather resistance.

Other monomers (C) having one ethylenically unsaturated group in one molecule include, for example, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl Alkyl (meth)acrylates such as (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate;
α-olefin ethylenically unsaturated monomers such as 1-propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.

Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , monomers having primary to tertiary amide groups such as diacetone (meth)acrylamide;
(meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamido-1,1-dimethylpropyl) ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl) ammonium chloride, and a monomer having a quaternary amino group such as trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride;
Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate Acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth)acrylate , n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexaethylene Examples include monomers having polyether chains such as glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate.

<Copolymerization composition of vinyl polymer>
The copolymerization composition of the vinyl polymer will be explained.
The tertiary amino group-containing monomer (A) is preferably used in an amount of 1% by mass or more based on the total amount of all monomers. Preferably it is 4% by mass or more, more preferably 10% by mass or more. Moreover, it is preferable to use it in an amount of 95% by mass or less. It is preferably 90% by mass or less, more preferably 50% by mass or less, particularly preferably 30% by mass or less. By setting it as the said range, the effect of long-term biofilm formation inhibitory ability is demonstrated.
The monomer (B) having a crosslinkable group is preferably used in an amount of 20% by mass or less based on the total amount of all monomers. Preferably it is 15% by mass or less, more preferably 10% by mass or less. By setting it as the said range, when a crosslinking agent is used together, the coating film which has an appropriate crosslinking density can be obtained.

<Oxidation>
Oxidation before polymerization and oxidation after polymerization will be explained. Oxidation before polymerization is performed using a solution containing the tertiary amino group-containing unsaturated monomer (A), and oxidation after polymerization is performed using a polymer obtained by polymerizing a monomer that essentially includes the tertiary amino group-containing unsaturated monomer (A). The tertiary amino group can be oxidized by adding an oxidizing agent to a solution containing the oxidizing agent and reacting at a temperature of 20° C. to 100° C. for 0.1 to 100 hours, preferably 1 to 50 hours.

As the oxidizing agent, an oxidizing agent such as peroxide or ozone is used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, and t-butyl hydroperoxide. Hydrogen peroxide is preferred, and is usually used in an aqueous solution. used in the form Although there are differences in degree, peroxide also functions as a radical generator, so in the case of vinyl polymers that use the tertiary amino group-containing unsaturated monomer (A) as an essential raw material, Oxidation is preferably performed after polymerization. Further, in the case of a urethane-based polymer described later, it is preferable to oxidize the polymer after polymerization so as to prevent side reactions from occurring.

Generally, the amount of the oxidizing agent used is 0.2 to 3 times the molar equivalent of the oxidizable functional group, that is, the tertiary amino group, and further 0.5 to 2 times the molar equivalent. More preferably, molar equivalents are used. The obtained polymer solution can also be used after treating residual peroxide by a known method. Specifically, reducing agent addition treatment, ion exchange treatment, activated carbon treatment, treatment using a metal catalyst, etc. can be mentioned.

The obtained polymer solution can be used as it is, but if necessary, the amine oxide group-containing polymer can also be isolated and used by known methods such as reprecipitation and solvent distillation. Further, the isolated amine oxide group-containing polymer can be further purified by reprecipitation, solvent washing, membrane separation, adsorption treatment, etc., if necessary.

As mentioned above, the vinyl polymer in the present invention includes those obtained by oxidizing the tertiary amino group-containing unsaturated monomer (A) and then polymerizing it with other monomers, and the tertiary amino group-containing unsaturated monomer (A) In addition to oxidized monomers obtained by polymerizing monomers that require Copolymerization using a reaction product of a group-containing unsaturated compound and an amine oxide group-containing compound such as hydroxyethyl-N,N-dimethylamine oxide can also be used.

<Amine oxide group content>
The amine oxide group imparts biofilm formation inhibiting properties to the polymer. The amine oxide group content in the vinyl polymer is preferably 0.25 to 5 mmol/g, more preferably 0.5 to 2 mmol/g. By setting the concentration to 0.25 to 5 mmol/g, the optimal ability to suppress biofilm formation can be maintained even when immersed in water for a long time.
When a vinyl polymer is obtained by polymerizing a monomer having an amine oxide group, the content of amine oxide groups in the vinyl polymer can be determined from the amount of the monomer having an amine oxide group used in the polymerization. On the other hand, when a polymer obtained after polymerizing a monomer that essentially includes a tertiary amino group-containing monomer is to be oxidized, it can be calculated using the above-mentioned formula 1.

<Mass average molecular weight (Mw)>
The weight average molecular weight of the vinyl polymer is 2,000 to 10,000,000, preferably 5,000 to 6,000,000, more preferably 10,000 to 600,000, and particularly preferably is between 10,000 and 100,000. When the molecular weight is 2,000 or more, cohesive force can be imparted, peeling from the coating substrate can be suppressed, and a long-term biofilm formation suppressing effect can be exhibited. Furthermore, when the molecular weight is 10,000,000 or less, the viscosity becomes appropriate, and thus the coating suitability is improved. Therefore, the weight average molecular weight of the vinyl polymer is limited within the above specific range.

As the mass average molecular weight of the vinyl polymer, a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) is used. The measuring device and measurement conditions are basically based on Condition 1 below, and Condition 2 is allowed depending on the solubility of the sample, etc. However, depending on the type of polymer, a suitable carrier (eluent) and a column suitable for the carrier may be selected and used. For other matters, please refer to JISK7252-1~4:2008. In addition, poorly soluble polymer compounds shall be measured at a concentration that allows them to be dissolved under the following conditions.
Furthermore, if it is difficult to measure the molecular weight of the vinyl polymer, the weight average molecular weight of the amine oxide precursor polymer can be set as the weight average molecular weight of the polymer (a). The weight average molecular weight of the amine oxide precursor polymer is determined by gel permeation chromatography (GPC) in terms of standard polystyrene, and the measurement device and measurement conditions are based on Condition 3 below.
(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
A combination of TOSOHTSKgelSuperHZ4000 and TOSOHTSKgelSuperHZ2000.
Carrier: Tetrahydrofuran Measurement temperature: 40℃
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 2)
Column: Two TOSOHTSKgelSuperAWM-H columns connected.
Carrier: 10mM LiBr/N-methylpyrrolidone Measurement temperature: 40°C
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<Crosslinking agent>
The biofilm formation inhibiting coating agent of the present invention can contain a crosslinking agent. By including a crosslinking agent, when the above-mentioned vinyl polymer has a crosslinkable group, it is possible to form crosslinks in the coating film and improve water resistance. The crosslinking agent that can be used in the present invention is selected from the group consisting of carboxyl groups, hydroxyl groups, epoxy groups, primary or secondary amino groups, and isocyanate groups in the monomer (B) for forming the vinyl polymer described above. Preferred examples include those having at least one functional group selected from epoxy groups, isocyanate groups, and aziridinyl groups, as well as metal chelate compounds and carbodiimide group-containing compounds. It will be done. These crosslinking agents can be used for the purpose of increasing the elastic modulus and resistance of the coating film, or for adjusting the adhesive strength.

[Crosslinking agent with epoxy group]
The crosslinking agent having an epoxy group used in the present invention is not particularly limited as long as it has two or more epoxy groups in one molecule. Examples of the crosslinking agent having a bifunctional epoxy group include ethylene glycol diglycidyl ether, polyethylene oxide diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, tetramethylene glycol diglycidyl ether, and polytetramethylene glycol diglycidyl ether. Ether, aliphatic epoxy compounds such as 1,6-hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, polybutadiene diglycidyl ether, bisphenol A type epoxy resin, bisphenol F type epoxy resin, bisphenol S type epoxy resin, biphenol type Aromatic epoxy compounds such as epoxy resins, dihydroxybenzophenone diglycidyl ether, resorcinol diglycidyl ether, hydroquinone diglycidyl ether, dihydroxyanthracene type epoxy resins, bisphenol fluorene glycidyl ether, N,N-diglycidylaniline, aromatic compounds described above Examples include hydrogenated epoxy compounds and alicyclic epoxy compounds such as hexahydrophthalic acid diglycidyl ester. Examples of the crosslinking agent having three or more epoxy groups include triglycidyl isocyanurate, trisphenol type epoxy compounds, tetrakisphenol type epoxy compounds, and phenol novolac type epoxy compounds.

[Crosslinking agent having isocyanate group]
The crosslinking agent having an isocyanate group used in the present invention is not particularly limited as long as it is a compound having two or more isocyanate groups in one molecule. Examples of the bifunctional isocyanate compound include 2,2'-diphenylmethane diisocyanate, 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, 2,6-tolylene diisocyanate, and hexane diisocyanate. Examples include methylene diisocyanate and isophorone diisocyanate.
Examples of the trifunctional isocyanate compound include a trimethylolpropane adduct of the diisocyanate described above, a biurette reacted with water, and a trimer having an isocyanurate ring.
Further, the isocyanate group in the crosslinking agent having an isocyanate group may be blocked or unblocked. The blocked isocyanate crosslinking agent used in the present invention may be a blocked isocyanate compound in which the isocyanate group in the isocyanate compound is blocked with ε-caprolactam, MEK oxime, cyclohexanone oxime, pyrazole, phenol, etc., and is particularly limited. It is not something that will be done.

[Crosslinking agent with aziridinyl group]
The aziridine compound used in the present invention is not particularly limited as long as it has two or more aziridine groups in one molecule. Examples of the aziridine compound include 2,2'-bishydroxymethylbutanol tris[3-(1-aziridinyl)propionate] and 4,4-bis(ethyleneiminocarbonylamino)diphenylmethane.

[Carbodiimide group-containing compound]
As the carbodiimide group-containing compound used in the present invention, the Carbodilite series manufactured by Nisshinbo Co., Ltd. can be used, including aqueous types such as V-02, V-04, V-06, and V-10, and V-01. , V-03, V-05, V-07, V-09, and other oil-based types.

[β-Hydroxyalkylamide group-containing compound]
In the present invention, compounds containing β-hydroxyalkylamide groups can also be used as crosslinking agents. The β-hydroxyalkylamide group-containing compound is not particularly limited as long as it is a compound containing a β-hydroxyalkylamide group in the molecule. Examples of the β-hydroxyalkylamide group-containing compound include various compounds including N,N,N',N'-tetrakis(hydroxyethyl)adipamide (PrimidXL-552 manufactured by M Chemie).

In the present invention, only one type of crosslinking agent may be used alone, or a plurality of them may be used in combination. The amount of crosslinking agent to be used may be determined by taking into account the type and number of moles of functional groups contained in the vinyl polymer, and is not particularly limited, but is usually based on 100 parts by mass of the vinyl polymer. It is used in a range of 0.1 parts by mass to 100 parts by mass. By blending in an amount smaller than the number of moles of functional groups contained in the vinyl polymer, it is possible to eliminate the concern that unreacted crosslinking agent will be liberated. Within this range, particularly excellent performance will be achieved in achieving the desired effects of inhibiting biofilm formation.

<Adjustment of coating agent for inhibiting biofilm formation>
The biofilm formation inhibiting coating agent of the present invention preferably contains 1 to 50% by mass, more preferably 5 to 30% by mass of the vinyl polymer based on 100% by mass of the coating agent. By setting the vinyl polymer content to 1% by mass or more, the effect of inhibiting biofilm formation due to amine oxide groups can be exhibited. Furthermore, the biofilm formation inhibiting coating agent of the present invention may contain components other than the vinyl polymer.

<Solvent>
The biofilm formation inhibiting coating agent of the present invention may contain a solvent as a component other than the vinyl polymer, or may contain two or more kinds in combination. The solvent can be appropriately selected from conventionally known solvents, taking into consideration the solubility of the vinyl polymer depending on the amount of amine oxide, printing conditions, etc. For example, if the amount of amine oxide in the vinyl polymer is large, water, alcohols such as methanol and ethanol, ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, methyl acetate and ethyl acetate, etc. Esters, dimethyl sulfoxide, acetonitrile, organic acids such as formic acid and acetic acid, and organic bases such as N,N-dimethylformamide can be selected. On the other hand, if the amount of amine oxide in the vinyl polymer is small, in addition to ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, esters such as methyl acetate and ethyl acetate, dimethyl sulfoxide, and acetonitrile, , halogen solvents such as dichloromethane and trichloromethane can be selected.

Furthermore, the biofilm formation inhibiting coating agent of the present invention may contain various additives as long as the effects of the present invention are not impaired.

<Biofilm formation suppression laminate>
The biofilm formation inhibiting laminate of the present invention has a coating film made of the biofilm formation inhibiting coating agent of the present invention on a base material. As a method for forming a coating film, various coating film forming methods (coating, printing, drying methods) can be selected depending on the substrate. Examples include, but are not limited to, various printing methods such as gravure and offset, as well as inkjet methods, spray methods, and dipping methods. Drying after coating may be performed as long as the solvent can be removed, and the drying temperature can be appropriately selected from the solvent contained in the biofilm formation inhibiting coating agent. Industrially, a temperature of 40 to 180°C for about 2 minutes is desirable. Furthermore, when the biofilm formation inhibiting coating agent of the present invention contains a crosslinking agent, it is preferable to provide a step for promoting the crosslinking reaction. Crosslinking conditions are generally 40-150° C. for 6-24 hours, but are not limited thereto. The thickness of the coating film made of the biofilm formation-inhibiting coating agent can be appropriately selected within a range that does not impair the effects of the present invention, and a thickness of 0.5 to 2 μm can sufficiently exhibit the effect.

<Base material>
The biofilm formation-inhibiting coating agent of the present invention can be applied to a wide range of fields where biofilm hazards are a concern. It can be suitably used on the surface of a substance that is expected to be exposed. Therefore, as the base material, any base material conventionally used in the above-mentioned applications can be used without limitation, and examples thereof include base materials made of materials such as plastic, glass, ceramics, and metal.

<Cell culture equipment treatment agent>
The cell culture equipment treatment agent has an amine oxide group and a mass average molecular weight of 2,000.
The above-mentioned vinyl polymer (A) is included.
In the present invention, the polymer having amine oxide groups can be obtained by the following two methods. That is, a monomer having an amine oxide group and another monomer can be polymerized to obtain a polymer having an amine oxide group. Alternatively, after obtaining a polymer having a tertiary amino group, which can be called a precursor functional group for an amine oxide group, the tertiary amino group can be reacted with an oxidizing agent to introduce an amine oxide group into the polymer. The latter method is preferred because it is less likely to cause a reaction. Note that reacting a tertiary amino group with an oxidizing agent is also referred to as "oxidation" hereinafter.

Specifically, the vinyl polymer preferably contains at least one of the structures represented by the above-mentioned general formulas 1 to 3, and among them, those containing the structure represented by general formula 1 are particularly preferable. The vinyl polymer is preferably a copolymer, and may be any of a block copolymer, random copolymer, and alternating copolymer.

Vinyl polymers having such a structure can be obtained in two ways, as described above. More preferably, the tertiary amino group-containing monomer (A) is oxidized and then polymerized with another monomer, or the tertiary amino group-containing monomer (A) and another monomer are polymerized and then oxidized. be.

<Tertiary amino group-containing monomer (A)>
Among the tertiary amino group-containing monomers (A) as precursors before oxidation, those for forming the structure of general formula 1 include:
For example, N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N, N-dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylamino Vinyl propionate, N,N-diethylaminovinyl propionate, N,N-dimethylacrylamide, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylamino Ethylstyrene, N,N-dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinylamine, or acid anhydrides containing unsaturated groups such as maleic anhydride, itaconic anhydride, and citraconic anhydride. , reaction products with N,N-dimethyl-1,3-propanediamine, etc., reaction of epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate with N,N-dimethyl-1,3-propanediamine, etc. products, etc.
Preferably it is a (meth)acrylate monomer, more preferably N,N-dialkylaminoethyl (meth)acrylate or N,N-dialkylaminopropyl (meth)acrylate.
In the present invention, "(meth)acrylic" refers to both "acrylic" and "methacrylic".

Examples of compounds for forming the structure of general formula 3 include 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-vinylpyridine, -Methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2- Examples include (t-butyl)-5-vinylpyridine.

Examples of substances for forming the structure of general formula 2 include 1-vinylimidazole, 2
-Methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4-(t-butyl)-1-vinylimidazole, and the like.

<Monomer (B) having a crosslinkable group>
From the viewpoint of improving the durability of the vinyl polymer, it is preferable that the vinyl polymer has at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group. Specifically, when obtaining a vinyl polymer, it is preferable to use, in addition to the monomer (A), a monomer (B) having at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group. . The crosslinkable group derived from the monomer (B) introduces a crosslinked structure into the polymer coating film by reacting with the crosslinking agent described below, thereby exhibiting excellent durability.

For example, a copolymer into which a carboxyl group has been introduced can be crosslinked with an epoxy compound, an aziridine compound, an oxetane, a carbodiimide compound, an amino compound, an isocyanate compound, or the like. The copolymer into which a hydroxyl group has been introduced can be crosslinked with a carbodiimide compound, an isocyanate compound, an amino compound, an alkoxylan compound, or the like.

The carboxyl group-containing monomer is not particularly limited as long as it has a carboxyl group in its structure; for example, (meth)acrylic acid, 2-carboxyethyl (meth)acrylate, or alkylene such as ethylene oxide or propylene oxide. Examples include alkylene oxide-added succinic acid (meth)acrylate having a carboxyl group at the end of which oxide is repeatedly added.

The hydroxyl group-containing monomer is not particularly limited as long as it has a hydroxyl group in its structure, and examples include 2-hydroxyethyl (meth)acrylate, 1-hydroxypropyl (meth)acrylate, and (meth)acrylic acid. 2-Hydroxypropyl, 3-hydroxypropyl (meth)acrylate, 1-hydroxybutyl (meth)acrylate, monofunctional glycerol (meth)acrylate, polylactone type (meth) having a hydroxyl group at the end by ring-opening addition of a lactone ring. ) Acrylic esters, alkylene oxide-added (meth)acrylic esters having a hydroxyl group at the end obtained by repeatedly adding alkylene oxides such as ethylene oxide or propylene oxide, and glucose ring-based (meth)acrylic esters.

From the viewpoint of durability, the vinyl polymer preferably has a carboxyl group as a crosslinkable group, and it is preferable to use a carboxyl group-containing monomer as the monomer (B) having a crosslinkable group constituting the polymer.

<Other monomers (C)>
When obtaining a vinyl polymer, other monomers (C) having one ethylenically unsaturated group in one molecule can be used in addition to the monomer (A). By introducing a structure based on the monomer (C), polarity and Tg can be appropriately controlled, and not only can the coating film have excellent coating properties and durability, but also solvent solubility and the like can be controlled.

Other monomers (C) having one ethylenically unsaturated group in one molecule include, for example, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, pentyl Alkyl (meth)acrylates such as (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate; α-olefinic ethylenically unsaturated monomers such as 1-propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.
Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid; monomers having phosphoric acid groups such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , diacetone (meth)acrylamide, and other monomers having primary to tertiary amide groups; (meth)acrylic acid dimethylaminoethylmethyl chloride salt, trimethyl-3-(1-(meth)acrylamide-1,1-dimethylpropyl) Monomers with quaternary amino groups such as ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl)ammonium chloride, and trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride. ; Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate; ) acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth) Acrylate, n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexa Examples include monomers having polyether chains such as ethylene glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate.

<Copolymer composition of vinyl polymer>
The copolymerization composition of the vinyl polymer will be explained.
The tertiary amino group-containing monomer (A) is preferably used in an amount of 1% by mass or more based on the total amount of all monomers. Preferably it is 4% by mass or more, more preferably 10% by mass or more. Moreover, it is preferable to use it in an amount of 95% by mass or less. It is preferably 90% by mass or less, more preferably 50% by mass or less, particularly preferably 30% by mass or less. Within the above range, long-term, low cytotoxicity, and excellent protein and cell adhesion prevention effects can be achieved. The monomer (B) having a crosslinkable group is preferably used in an amount of 20% by mass or less based on the total amount of all monomers. Preferably it is 15% by mass or less, more preferably 10% by mass or less. By setting it as the said range, when a crosslinking agent is used together, the coating film which has an appropriate crosslinking density can be obtained.

<Oxidation>
Oxidation before polymerization and oxidation after polymerization will be explained. Oxidation before polymerization is performed using a solution containing the tertiary amino group-containing unsaturated monomer (A), and oxidation after polymerization is performed using a polymer obtained by polymerizing monomers that essentially include the tertiary amino group-containing unsaturated monomer (A). The tertiary amino group can be oxidized by adding an oxidizing agent to a solution containing the oxidizing agent and reacting at a temperature of 20° C. to 100° C. for 0.1 to 100 hours, preferably 1 to 50 hours.

As the oxidizing agent, an oxidizing agent such as peroxide or ozone is used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, and t-butyl hydroperoxide. Hydrogen peroxide is preferred, and is usually used in an aqueous solution. used in the form Although there are differences in degree, peroxide also functions as a radical generator, so in the case of vinyl polymers that use the tertiary amino group-containing unsaturated monomer (A) as an essential raw material, Oxidation is preferably performed after polymerization. Further, in the case of a urethane-based polymer described later, it is preferable to oxidize the polymer after polymerization so as to prevent side reactions from occurring.

Generally, the amount of the oxidizing agent used is 0.2 to 3 times the molar equivalent of the oxidizable functional group, that is, the tertiary amino group, and further 0.5 to 2 times the molar equivalent. More preferably, molar equivalents are used. The obtained polymer solution can also be used after treating residual peroxide by a known method. Specifically, reducing agent addition treatment, ion exchange treatment, activated carbon treatment, treatment using a metal catalyst, etc. can be mentioned. The obtained polymer solution can be used as it is, but if necessary, the amine oxide group-containing polymer can also be isolated and used by known methods such as reprecipitation and solvent distillation. Further, the isolated amine oxide group-containing polymer can be further purified by reprecipitation, solvent washing, membrane separation, adsorption treatment, etc., if necessary.

As mentioned above, the vinyl polymer in the present invention includes those obtained by oxidizing the tertiary amino group-containing unsaturated monomer (A) and then polymerizing it with other monomers, and the tertiary amino group-containing unsaturated monomer (A) In addition to oxidized monomers obtained by polymerizing monomers that require Copolymerization using a reaction product of a group-containing unsaturated compound and an amine oxide group-containing compound such as hydroxyethyl-N,N-dimethylamine oxide can also be used.

<Amine oxide group content>
The amine oxide group imparts an anti-cell adhesion effect to the polymer. The amine oxide group content in the vinyl polymer is preferably 0.25 to 5 mmol/g, more preferably 0.5 to 5 mmol/g. By setting the concentration to 0.25 to 5 mmol/g, the cell adhesion prevention effect can be maintained even when immersed in water for a long time.
When a vinyl polymer is obtained by polymerizing a monomer having an amine oxide group, the content of amine oxide groups in the vinyl polymer can be determined from the amount of the monomer having an amine oxide group used in the polymerization. On the other hand, when a polymer obtained after polymerizing a monomer that essentially includes a tertiary amino group-containing monomer is to be oxidized, it can be calculated using the above-mentioned formula 1.

<Mass average molecular weight (Mw)>
The weight average molecular weight of the vinyl polymer (A) is 2,000 or more, preferably 2,000 to 10,000,000, more preferably 5,000 or more, and still more preferably 10,000 or more. and particularly preferably 100,000 or more. When the molecular weight is 2,000 or more, it is possible to impart cohesive force to the coating film, suppress peeling from the coating substrate, and exhibit a long-term cell adhesion prevention effect. In addition, when it is 10,000,000 or less, the viscosity becomes appropriate, which improves coating suitability, which is preferable. More preferably, it is 500,000 or less.

As the mass average molecular weight of the vinyl polymer, a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) is used. The measuring device and measurement conditions are basically based on Condition 1 below, and Condition 2 is allowed depending on the solubility of the sample, etc. However, depending on the type of polymer, a suitable carrier (eluent) and a column suitable for the carrier may be selected and used. For other matters, please refer to JISK7252-1~4:2008. In addition, poorly soluble polymer compounds shall be measured at a concentration that allows them to be dissolved under the following conditions. Furthermore, if it is difficult to measure the molecular weight of the vinyl polymer, the weight average molecular weight of the amine oxide precursor polymer can be set as the weight average molecular weight of the vinyl polymer. The weight average molecular weight of the amine oxide precursor polymer is determined by gel permeation chromatography (GPC) in terms of standard polystyrene, and the measurement device and measurement conditions are based on Condition 3 below.

(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
A combination of TOSOHTSKgelSuperHZ4000 and TOSOHTSKgelSuperHZ2000.
Carrier: Tetrahydrofuran Measurement temperature: 40℃
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL

(Condition 2)
Column: Two TOSOHTSKgelSuperAWM-H columns connected.
Carrier: 10mM LiBr/N-methylpyrrolidone Measurement temperature: 40°C
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL

(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<Crosslinking agent>
The cell culture equipment treatment agent of the present invention can further contain a crosslinking agent. By including a crosslinking agent, when the above-mentioned vinyl polymer has a crosslinkable group, it is possible to form crosslinks in the coating film and improve durability (water resistance).
The crosslinking agent that can be used in the present invention is preferably one that reacts with at least one crosslinkable group selected from the group consisting of carboxyl groups and hydroxyl groups in the monomer (B) for forming the vinyl polymer described above. For example, examples of the crosslinking agent for the vinyl polymer (A) into which a carboxyl group has been introduced include isocyanate compounds, aziridine compounds, carbodiimide compounds, and amino compounds. Further, examples of the crosslinking agent for the vinyl polymer (A) into which a hydroxyl group has been introduced include carbodiimide compounds, isocyanate compounds, amino compounds, alkoxylan compounds, and the like. These crosslinking agents can be used for the purpose of increasing the elastic modulus and resistance of the coating film, or for adjusting the adhesive strength to the substrate. A carbodiimide compound and an isocyanate compound will be explained below as examples of preferable crosslinking agents.

[Carbodiimide group-containing compound (carbodiimide compound)]
Examples of carbodiimide group-containing compounds include dicarbodiimides such as N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-di-2,6-diisopropylphenylcarbodiimide, and poly(1,6-hexane). methylenecarbodiimide), poly(4,4'-methylenebiscyclohexylcarbodiimide), poly(1,3-cyclohexylenecarbodiimide), poly(1,4-cyclohexylenecarbodiimide), poly(4,4'-dicyclohexylmethanecarbodiimide) , poly(4,4'-diphenylmethanecarbodiimide), poly(3,3'-dimethyl-4,4'-diphenylmethanecarbodiimide), poly(naphthalenecarbodiimide), poly(p-phenylenecarbodiimide), poly(m-phenylenecarbodiimide) ), poly(tolylcarbodiimide), poly(diisopropylcarbodiimide), poly(methyl-diisopropylphenylenecarbodiimide), poly(1,3,5-triisopropylbenzene) polycarbodiimide, poly(1,3,5-triisopropylbenzene) Examples include polycarbodiimides such as polycarbodiimide, poly(1,5-diisopropylbenzene)polycarbodiimide, poly(triethylphenylenecarbodiimide), and poly(triisopropylphenylenecarbodiimide).

Examples of commercially available carbodiimide group-containing compounds include "Carbodilite" (registered trademark) manufactured by Nisshinbo Holdings, Inc. and "Stabaxol (registered trademark)" manufactured by Rein Chemie.

[Crosslinking agent having isocyanate group (isocyanate compound)]
The crosslinking agent having isocyanate groups used in the present invention is not particularly limited as long as it is a compound having two or more isocyanate groups in one molecule.
Examples of the bifunctional isocyanate compound include 2,2'-diphenylmethane diisocyanate, 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, 2,6-tolylene diisocyanate, and hexane diisocyanate. Examples include methylene diisocyanate and isophorone diisocyanate.
Examples of the trifunctional isocyanate compound include a trimethylolpropane adduct of the diisocyanate described above, a biurette reacted with water, and a trimer having an isocyanurate ring.
Further, the isocyanate group in the crosslinking agent having an isocyanate group may be blocked or unblocked. The blocked isocyanate crosslinking agent used in the present invention may be a blocked isocyanate compound in which the isocyanate group in the isocyanate compound is blocked with ε-caprolactam, MEK oxime, cyclohexanone oxime, pyrazole, phenol, etc., and is particularly limited. It is not something that will be done.

In the present invention, only one type of crosslinking agent may be used alone, or a plurality of them may be used in combination. The amount of crosslinking agent to be used may be determined by taking into account the type and number of moles of functional groups contained in the vinyl polymer, and is not particularly limited, but is usually based on 100 parts by mass of the vinyl polymer. It is used in a range of 0.1 parts by mass to 100 parts by mass. By blending in an amount smaller than the number of moles of functional groups contained in the vinyl polymer, it is possible to eliminate the concern that unreacted crosslinking agent will be liberated. Within this range, the intended cell culture equipment treatment agent exhibits particularly excellent performance in each effect.

<Preparation of cell culture equipment treatment agent>
The cell culture equipment treatment agent of the present invention preferably contains 50% by mass or less of the vinyl polymer, more preferably 0.5 to 10% by mass, based on 100% by mass of the cell culture equipment treatment agent. By setting the vinyl polymer content to 0.5% by mass or more, the effect of suppressing cell adhesion due to amine oxide groups can be exhibited. Furthermore, the cell culture equipment treatment agent of the present invention may contain components other than the vinyl polymer.

<Solvent>
The cell culture equipment treatment agent of the present invention may contain a solvent as a component other than the vinyl polymer, or may contain two or more kinds in combination. The solvent can be appropriately selected from conventionally known solvents, taking into consideration the solubility of the vinyl polymer depending on the amount of amine oxide, film forming conditions, and the like.
For example, if the amount of amine oxide in the vinyl polymer is large, water, alcohols such as methanol and ethanol, ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, methyl acetate and ethyl acetate, etc. Esters, dimethyl sulfoxide, acetonitrile, organic acids such as formic acid and acetic acid, and organic bases such as N,N-dimethylformamide can be selected. On the other hand, if the amount of amine oxide in the vinyl polymer is small, in addition to ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, esters such as methyl acetate and ethyl acetate, dimethyl sulfoxide, and acetonitrile, , halogen solvents such as dichloromethane and trichloromethane can be selected.

<Other additives, etc.>
The cell culture equipment treatment agent of the present invention may contain various additives as long as the effects of the present invention are not impaired, such as surfactants, isotonic agents, chelating agents, pH adjusters, and buffers. , thickeners, stabilizers, proteolytic enzymes, pharmacologically active ingredients, physiologically active ingredients, and various additives listed in the Dictionary of Pharmaceutical Excipients, etc., may be included within the range that does not impair the effects of the present invention. . These may contain one type alone or two or more types.

For example, a pH adjuster may be included to adjust the ion balance. Examples of the pH adjuster include acidic or basic compounds. Examples of acidic pH adjusters include carboxylic acids, inorganic acids and sulfonic acids, and examples of basic pH adjusters include hydroxides, alkoxides, nitrogen-containing compounds other than primary and secondary amines. , basic carbonates, and basic phosphates. The pH value of the cell culture equipment treatment agent is about 4.0 to 9.0, preferably about 6.0 to 8.0, and is preferably adjusted to around 7.0 before use.

Further, examples of isotonic agents include sugars, polyalcohols (mannitol, sorbitol, etc.), sodium chloride, and the like. Thickeners and stabilizers include synthetic organic polymer compounds such as polyvinyl alcohol, poly-N-vinylpyrrolidone, polyethylene glycol, polypropylene glycol, and polyacrylamide, and cellulose derivatives such as hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose. , sodium carboxymethyl starch, starch derivatives such as hydroxyethyl starch, chondroitin sulfate, hyaluronate, and the like.

<Cell culture equipment>
The cell culture equipment of the present invention has a cell adhesion prevention film (sometimes also referred to as a coating film) on a base material containing a vinyl polymer containing an amine oxide group and having a mass average molecular weight of 2,000 or more. and a step of forming a cell adhesion prevention film containing a vinyl polymer having an amine oxide group and a mass average molecular weight of 2,000 or more on a base material, specifically, amine oxide It can be manufactured by including a step of coating a cell culture equipment treatment agent containing a vinyl polymer containing a group and having a mass average molecular weight of 2,000 or more.

The method for forming a cell adhesion prevention film, that is, the step of coating a cell culture equipment treatment agent containing a vinyl polymer containing an amine oxide group and having a mass average molecular weight of 2,000 or more, is carried out by a known method. be able to. For example, a coating film can be formed by applying a heat treatment or the like after coating the cell culture equipment treatment agent using a known method. Note that it can also be cured using a crosslinking agent.

The coating method can be appropriately selected depending on the substrate, and specific examples thereof include spray coating, dip coating, flow coating, inkjet coating, brush coating, sponge coating, and the like. Furthermore, various printing methods such as gravure printing, offset printing, and silk screen printing may be used. The heat treatment temperature after coating can be appropriately selected depending on the boiling point of the solvent contained and the crosslinking temperature of the crosslinking agent, but industrially it is preferably 40 to 180°C. Furthermore, when the present composition contains a crosslinking agent, it is preferable to provide a step for promoting the crosslinking reaction. Crosslinking conditions are generally 40-150° C. for 6-24 hours, but are not limited thereto.

<Base material>
The substrate of the present invention is not particularly limited as long as it can be used for cell culture equipment. Specifically, (meth)acrylic resin, styrene resin, polycarbonate resin, polyolefin resin, polystyrene, polyethylene, polyvinyl chloride, polypropylene, polyester, polyvinyl acetate, vinyl-acetate copolymer, styrene-methylmeth Acrylate copolymer, acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, nylon, polymethylpentene, silicone resin, amino resin, polysulfone, polyethersulfone, polyetherimide, fluororesin, and Examples include one or more resins selected from the group consisting of polyimide resin materials, as well as glass substrates.

<Silicone base material>
The silicone base material is not particularly limited as long as it contains a silicone resin.
Examples of the silicone resin include those having a main chain of polyorganosiloxane such as polydimethylsiloxane, polyphenylmethylsiloxane, and polydiphenylsiloxane, and preferably polydimethylsiloxane. Further, specific examples of such a silicone base material include a silicone-containing medical device, a silicone base material having a microchannel, and preferably a silicone-containing microchannel device.

The above-mentioned medical device refers to a medical structure used in a living body, and such a structure can be used by being implanted in the body or used in the body (without being implanted). It is broadly divided into Note that the size and length of the medical device are not particularly limited, and include those having minute circuits and those capable of detecting a minute amount of sample.
Structures to be implanted into the body include, for example, functional auxiliary devices such as cardiac pacemakers to supplement the functions of living organisms suffering from diseases; devices for detecting abnormalities in living organisms such as implantable biochips; Examples include medical devices such as implants, bone fixing materials, sutures, and artificial blood vessels. In addition, examples of structures used in the body (without being implanted) include catheters, gastrocameras, and the like.

Examples of the microchannel device include microanalysis devices such as microreaction devices (specifically, microreactors and microplants), integrated nucleic acid analysis devices, microelectrophoresis devices, and microchromatography devices; Microdevices for analytical sample preparation such as liquid chromatography; physicochemical processing devices used for extraction, membrane separation, dialysis, etc.; environmental analysis chips, clinical analysis chips, genetic analysis chips (DNA chips), protein analysis chips (proteome chips) , microchannel chips such as sugar chain chips, chromatography chips, cell analysis chips, and pharmaceutical screening chips. Among these, microchannel chips are preferred.

The microchannel is a part through which a small amount of sample (preferably a liquid sample) flows, and the width and depth of the channel are not particularly limited, but are usually about 0.1 μm to 1 mm, Preferably it is 10 μm to 800 μm. Note that the channel width and depth of the microchannel may be the same over the entire length of the channel, or may be partially different in size and shape.

The silicone base material may be subjected to plasma treatment, UV ozone treatment, internal infiltrating agent treatment, etc. The silicone base material that has undergone these treatments has a hydrophilic surface, which may make it difficult for polymers to adsorb, but silicone base materials that contain amine oxide groups and have a mass average molecular weight of 2, A vinyl-based polymer having a molecular weight of 000 or more also adsorbs to silicone substrates that have been subjected to such plasma treatment, thereby imparting excellent hydrophilicity and the effect of suppressing adsorption of biological samples.

<Medical device>
The medical device is not particularly limited as long as it is a medical device, and specific examples include blood bags, urine collection bags, blood transfusion sets, sutures, drain tubes, various catheters, blood accesses, blood circuits, artificial blood vessels, Examples include artificial kidneys, artificial heart-lung machines, artificial valves, plasmapheresis membranes, various adsorbents, CAPDs, IABPs, pacemakers, artificial joints, artificial femoral heads, dental materials, and various shunts.
The material and shape of the medical device are not particularly limited, and materials include polyolefin, modified polyolefin, polyether, polyurethane, polyamide, polyimide, polyester such as polyethylene terephthalate, polytetrafluoroethylene, polyvinyl chloride, and copolymers thereof. Examples include various polymeric materials such as metals, ceramics, carbon, and composite materials thereof. Further, the shape is not particularly limited, and may be smooth or porous.

<Cell culture medium additives>
The cell culture medium additive of the present invention contains a vinyl polymer (A) having an amine oxide group and having a mass average molecular weight of 2,000 or more. By including the vinyl polymer (A), the cells do not adhere to the base material, and exhibit the effect of forming a three-dimensional cell aggregate with a good ratio of viable cells.
Moreover, the cell culture medium additive of the present invention can efficiently produce antibodies by containing the vinyl polymer (A). In addition, the generation of antibody-producing cell aggregates is suppressed, making it easier to purify the desired antibody.

<Vinyl polymer (A)>
The vinyl polymer (A) of the present invention only needs to have an amine oxide group and a mass average molecular weight of 2,000 or more, and conventionally known polymers can be used, or two or more types can be used in combination. Good too. By having an amine oxide group, it exhibits excellent cell aggregate forming properties.

In the present invention, the vinyl polymer having amine oxide groups can be obtained by the following two methods.
(1) A method of obtaining a copolymer of a monomer having an amine oxide group and other monomers.
(2) A method of obtaining a vinyl polymer having a tertiary amino group, which can be called a precursor functional group for an amine oxide group, and then obtaining a reaction product of the tertiary amino group and an oxidizing agent.
(3) A method of obtaining a copolymer of a reaction product monomer of a monomer having a tertiary amino group and an oxidizing agent (amine oxide group-containing monomer) and other monomers.
An amine oxide group can be introduced by reacting an oxidizing agent (hereinafter also referred to as an oxidizing agent) with a tertiary amino group. This reaction is also referred to as "oxidation" hereinafter. Method (2) is preferred in that side reactions are less likely to occur.

Specifically, the vinyl polymer preferably contains at least one of the structures represented by the above-mentioned general formulas 1 to 3, and among them, those containing the structure represented by general formula 1 are particularly preferable. When the amine oxide group is represented by the following formula, suitable water solubility can be exhibited.
The vinyl polymer is preferably a copolymer, and may be a block copolymer, random copolymer, or alternating copolymer.

Vinyl polymers having such a structure can be obtained in three ways, as described above. More preferred is the method (2) described above, in which the tertiary amino group-containing monomer (a) and other monomers are polymerized and then oxidized. Other monomers preferably include a monomer (c) having an alkyl group having 4 to 18 carbon atoms.

<Tertiary amino group-containing monomer (a)>
Among the tertiary amino group-containing monomers (a) as precursors before oxidation,
For forming the structure of general formula 1, for example,
N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N,N- Dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylaminopropionic acid Vinyl, N,N-diethylaminopropionate vinyl, N,N-dimethylacrylamide, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylaminoethylstyrene , N,N-dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinylamine, or an acid anhydride containing an unsaturated group such as maleic anhydride, itaconic anhydride, citraconic anhydride; , N-dimethyl-1,3-propanediamine, etc., and reaction products of epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propanediamine, etc. etc.
Preferably, it is a (meth)acrylate monomer, more preferably N,N-dialkylaminoethyl (meth)acrylate or N,N-dialkylaminopropyl (meth)acrylate.
In the present invention, "(meth)acrylic" refers to both "acrylic" and "methacrylic."

For forming the structure of general formula 3, for example,
2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine , 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2-(t-butyl)-5-vinylpyridine, and the like.

For forming the structure of general formula 2, for example,
Examples include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4-(t-butyl)-1-vinylimidazole, and the like.

<Other monomers>
When obtaining the vinyl polymer (A), other monomers having one ethylenically unsaturated group in one molecule can be used in addition to the tertiary amino group-containing monomer (a). By introducing a structure based on other monomers, polarity and Tg can be appropriately controlled, and solvent solubility and the like can be controlled.
As other monomers, it is preferable to use a monomer (c) having an alkyl group having 4 to 18 carbon atoms, from the viewpoint of cell aggregate formation and the ability to inhibit adsorption of cell aggregates.

[Monomer (c) having an alkyl group having 4 to 18 carbon atoms]
The monomer (c) having an alkyl group having 4 to 18 carbon atoms is preferably a monomer having one ethylenically unsaturated group and an alkyl group having 4 to 18 carbon atoms in one molecule. Since the vinyl polymer (A) has an alkyl group having 4 to 18 carbon atoms, the polarity can be controlled and the surrounding environment can be appropriately controlled.

The monomer (c) is not particularly limited, but for example,
Butyl (meth)acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate, Alkyl (meth)acrylates such as stearyl (meth)acrylate, cetyl (meth)acrylate, cyclohexyl (meth)acrylate;
Examples include α-olefin ethylenically unsaturated monomers such as 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.

[Monomer (d) other than (a) and (c)]
Furthermore, when obtaining a vinyl polymer, a monomer (c) other than the above-mentioned monomers (a) and (c) may be used. Monomers (d) other than (a) and (c) can be copolymerized with monomers (a) and (c), and have one ethylenically unsaturated group in one molecule other than monomers (a) and (c). It is preferable that the monomer has the following.

The monomer (d) is not particularly limited, but
Methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, etc. Alkyl (meth)acrylate, isobonyl (meth)acrylate, methoxyethyl (meth)acrylate, ethoxyethyl (meth)acrylate, propoxyethyl (meth)acrylate , acrylic ester (meth)acrylates such as butoxyethyl (meth)acrylate, ethoxypropyl (meth)acrylate;
Aromatic ester (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, phenoxyethyl (meth)acrylate;
Allyl (meth)acrylate, 1-methylallyl (meth)acrylate, 2-methylallyl (meth)acrylate, 1-butenyl (meth)acrylate, 2-butenyl (meth)acrylate, 3-(meth)acrylate Butenyl, 1,3-methyl-3-butenyl (meth)acrylate, 2-chlorallyl (meth)acrylate, 3-chlorallyl (meth)acrylate, o-allylphenyl (meth)acrylate, (meth)acrylic acid 2-(allyloxy)ethyl,
Allyl lactyl (meth)acrylate, citronellyl (meth)acrylate, geranyl (meth)acrylate, rhodinyl (meth)acrylate, cinnamyl (meth)acrylate, diallyl maleate, diallylitaconic acid, vinyl (meth)acrylate, Ethylenically unsaturated group-containing (meth)acrylic acid esters such as vinyl crotonate, vinyl oleate, vinyl linolenate, 2-(2'-vinyloxyethoxy)ethyl (meth)acrylate;
Perfluoromethylmethyl (meth)acrylate, perfluoroethylmethyl (meth)acrylate, 2-perfluorobutylethyl (meth)acrylate, 2-perfluorohexylethyl (meth)acrylate, 2-perfluorooctylethyl (meth)acrylate , 2-perfluoroisononylethyl (meth)acrylate, 2-perfluorononylethyl (meth)acrylate, 2-perfluorodecylethyl (meth)acrylate, perfluoropropylpropyl (meth)acrylate, perfluorooctylpropyl (meth)acrylate ) acrylate, perfluorooctyl amyl (meth)acrylate, perfluorooctyl undecyl (meth)acrylate, etc. (meth) such as perfluoroalkyl group-containing ethylenically unsaturated monomers having a perfluoroalkyl group having 1 to 20 carbon atoms. Acrylate monomer;
2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-(meth)acryloyloxyethyl-2-hydroxyethylphthalate, glycerol mono(meth)acrylate , 4-hydroxyvinylbenzene, 1-ethynyl-1-cyclohexanol, allyl alcohol, and other hydroxyl group-containing monomers;
Maleic acid, fumaric acid, itaconic acid, citraconic acid, or alkyl or alkenyl monoesters thereof, β-(meth)acryloxyethyl phthalate monoester, β-(meth)acryloxyethyl isophthalate monoester, terephthalic acid A monomer having a carboxylic acid group, such as β-(meth)acryloxyethyl monoester, β-(meth)acryloxyethyl succinate monoester, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, or its anhydride;
Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , monomers having primary to tertiary amide groups such as diacetone (meth)acrylamide;
(meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamido-1,1-dimethylpropyl) ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl) ammonium chloride, and a monomer having a quaternary amino group such as trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride;
Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate Acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth)acrylate , n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexaethylene Monomers having polyether chains such as glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate;
(Meth)acrylate monomers with a polymer structure in the side chain, such as ethylenically unsaturated compounds with polyester chains such as lactone-modified (meth)acrylate; styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allyl Aromatic vinyl monomers such as benzene and ethynylbenzene; ethylenically unsaturated monomers containing nitrile groups such as (meth)acrylonitrile; vinyl acetate, vinyl butyrate, vinyl propionate, vinyl hexanoate, vinyl caprylate, vinyl laurate, palmitic acid Fatty acid vinyl compounds such as vinyl and vinyl stearate; vinyl ether ethylenically unsaturated monomers such as butyl vinyl ether and ethyl vinyl ether;
Allyl monomers such as allyl acetate and allyl cyanide; Vinyl monomers such as vinyl cyanide, vinyl cyclohexane, and vinyl methyl ketone; Ethynyl monomers such as acetylene and ethynyltoluene; perfluorobutyl ethylene, perfluorohexyl ethylene, perfluorooctyl ethylene, and Examples include monomers having ethylenically unsaturated bonds that are not (meth)acrylates, such as ethylenically unsaturated compounds containing perfluoroalkyl groups such as fluorodecyl ethylene and perfluoroalkyl groups such as alkylenes.

<Copolymer composition of vinyl polymer (A)>
The copolymer composition of polymer (a) will be explained.
The tertiary amino group-containing monomer (a) is preferably contained in an amount of 20 to 99.9% by mass, more preferably 65 to 85% by mass, based on the total amount of all monomers. By setting it as the said range, especially excellent cell aggregate formation property will be demonstrated.
The monomer (c) having an alkyl group having 4 to 18 carbon atoms is preferably contained in an amount of 0.1 to 80% by mass, more preferably 12 to 40% by mass, based on the total amount of all monomers.
The other monomer (d) is preferably contained in an amount of 0 to 79.9% by mass, more preferably 0 to 50% by mass, particularly preferably 0 to 25% by mass, based on the total amount of all monomers.

<Oxidation>
The tertiary amine group derived from monomer (a) can be converted into an amine oxide group by using various oxidizing agents. Thereby, an amine oxide group can be introduced into the vinyl polymer (A). The oxidation may be performed either before or after the polymerization of the vinyl polymer (A), and the oxidation before the polymerization is performed by adding the tertiary amino group-containing monomer (a) to the solution containing the tertiary amino group-containing monomer (a). In this method, an oxidizing agent is added to a solution containing a vinyl polymer obtained by polymerizing a monomer containing a tertiary amino group-containing monomer (a), and the mixture is heated at a temperature of 20° C. to 100° C. for 0.1 to 100 hours, preferably. The tertiary amino group can be oxidized by reacting for 1 to 50 hours.

As the oxidizing agent, an oxidizing agent such as peroxide or ozone is used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, t-butyl hydroperoxide, etc. Hydrogen peroxide is preferred, and is usually used in an aqueous solution. used in the form Although there are differences in degree, peroxide also functions as a radical generator, so in the case of vinyl polymers that use tertiary amino group-containing unsaturated monomer (A) as an essential raw material, Oxidation is preferably performed after polymerization.

Generally, the amount of the oxidizing agent used is 0.2 to 3 times the molar equivalent of the oxidizable functional group, that is, the tertiary amino group, and further 0.5 to 2 times the molar equivalent. More preferably, molar equivalents are used. The obtained polymer solution can also be used after treating residual peroxide by a known method. Specifically, reducing agent addition treatment, ion exchange treatment, activated carbon treatment, treatment using a metal catalyst, etc. can be mentioned. The obtained polymer solution can be used as it is, but if necessary, the amine oxide group-containing polymer can also be isolated and used by known methods such as reprecipitation and solvent distillation. Further, the isolated amine oxide group-containing polymer can be further purified by reprecipitation, solvent washing, membrane separation, adsorption treatment, etc., if necessary.

As mentioned above, the vinyl polymer in the present invention includes a tertiary amino group-containing monomer (a) that is oxidized and then polymerized with other monomers, and a tertiary amino group-containing monomer (a) that is essential. In addition to those obtained by polymerizing monomers and oxidizing them after obtaining a polymer, examples of monomers include epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and isocyanate group-containing unsaturated compounds such as 2-isocyanate ethyl (meth)acrylate. Copolymerization using a reaction product of a compound and an amine oxide group-containing compound such as hydroxyethyl-N,N-dimethylamine oxide can also be used.

<Amine oxide group content>
Amine oxide groups have the effect of appropriately controlling the surrounding environment of proteins and suppressing functional deactivation. The amine oxide group content in the polymer (A) is preferably 1 to 10 mmol/g, more preferably 2 to 8 mmol/g. When the content of amine oxide groups is within the above range, suitable water solubility is exhibited, polarity is controlled, and the surrounding environment can be appropriately controlled.

When obtaining the polymer (A) by polymerizing monomers having amine oxide groups, the content of amine oxide groups in the vinyl polymer (A) can be determined from the amount of monomers having amine oxide groups used in the polymerization. I can do it. On the other hand, in the case of oxidizing a polymer obtained after polymerizing a monomer that essentially includes the tertiary amino group-containing monomer (a), it can be calculated using the above-mentioned formula 1.

<Mass average molecular weight (Mw)>
The weight average molecular weight of the vinyl polymer (A) is preferably 2,000 to 10,000,000, more preferably 5,000 or more, still more preferably 10,000 or more, and particularly preferably 20,000 or more. ,000 or more. More preferably, it is 500,000 or less, particularly preferably 200,000 or less. When the molecular weight is within the above range, the thickening effect can be reduced and problems such as gelation can be prevented. In addition, handling such as pipetting becomes easier.

(Method for measuring mass average molecular weight (Mw))
The mass average molecular weight of the vinyl polymer (A) was determined by gel permeation chromatography (GPC) in terms of standard pullulan. The following Condition 1 was used as the measuring device and measurement conditions. For other matters, JISK7252-1 to 4:2008 was referred to. Note that poorly soluble polymer compounds were measured at soluble concentrations under the following conditions. Furthermore, if it is difficult to measure the molecular weight of the vinyl polymer (A), the weight average molecular weight of the amine oxide precursor polymer can be set as the weight average molecular weight of the vinyl polymer (A). The mass average molecular weight of the amine oxide precursor polymer was determined by gel permeation chromatography (GPC) in terms of standard polystyrene, and the measurement device and measurement conditions were based on Condition 2 below.

(Condition 1)
Column: OHpak SB-G,
3 OHpak SB-806M HQ and
OHpak SB-802.5 HQ connected.
Carrier: 1/15 mol/L pH 7.0 phosphate buffer (phosphate buffer powder 1/15 mol/L pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 1 L of ion-exchanged water. thing)
Carrier flow rate: 1.0mL/min
Sample concentration: 0.5% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL

(Condition 2)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<Method for producing cell aggregates>
In the method for producing a cell aggregate according to this embodiment, a cell culture medium additive containing the above-mentioned vinyl polymer (A) is added to a cell culture medium. By adding the vinyl polymer (A) to the culture medium, cells no longer adhere to the culture vessel. Therefore, in the manufacturing method according to the present embodiment, cell aggregates can be easily formed by static culture.

In the present invention, the amount of the vinyl polymer (A) added is preferably in the range of 0.01% by mass or more and 5% by mass or less based on the cell culture medium. Cell aggregates can be particularly well formed when the amount is more than 0.05% by mass. Further, from the viewpoint of solubility in the medium, the amount of the vinyl polymer (A) added is preferably 1% by mass or less with respect to the medium.

Animal cells to which the method for producing cell aggregates according to the present embodiment can be applied are not particularly limited. Examples of such animal cells include cells derived from mammals such as humans, mice, and rats.

The cell culture medium that can be used in this embodiment is not particularly limited. Examples of such a medium include various commercially available media (αMEM, MEM, DMEM, IMDEM, RPMI1640, DMEM/F12, etc.) and combinations thereof.

The cell culture medium contains various growth factors (epidermal growth factor, insulin-like growth factor, nerve growth factor, hepatocyte growth factor, vascular endothelial growth factor, basic fibroblast growth factor, transferrin, steroid hormones) as needed. , 2-mercaptoethanol, etc.), various animal serums (foetal bovine serum (FBS), bovine serum, etc.), serum substitutes, etc. are added.

As a culture container applicable to this embodiment, a general culture container can be adopted. Examples of such culture vessels include multiwell plates, petri dishes, bag-like containers, and culture flasks. Examples of materials for these culture containers include polystyrene.

The culture conditions in this embodiment may be normal animal cell culture conditions, such as a 5% CO ₂ atmosphere and a temperature of 37°C.

<Method for producing antibodies>
The method for producing an antibody according to this embodiment includes culturing antibody-producing cells using a cell culture medium containing an antibody-producing cell culture medium additive containing the vinyl polymer (A) described above.
The amount of vinyl polymer (A) added is preferably in the range of 0.01% by mass or more and 5% by mass or less based on the cell culture medium, and the amount of vinyl polymer (A) added is When the amount is more than 0.05% by mass, the generation of antibody-producing cell aggregates can be particularly suppressed. Further, from the viewpoint of solubility in the medium, the amount of the vinyl polymer (A) added is preferably 1% by mass or less with respect to the medium.

The animal cells cultured in the culture method of the present invention are not particularly limited, and the cells are not particularly limited as long as they can be used for antibody production, such as CHO cells, BHK cells, HepG2 cells, rodent myeloma cells (e.g. Examples include mouse myeloma cells such as SP2/O cells and NSO cells), hybridomas, insect cells, and transformed cells in which foreign genes are introduced into these cells, such as CHO cells, SP2/O cells, or NSO cells. Hybridomas etc. obtained by cell fusion of cells etc. can be employed as antibody producing cells.

Antibodies produced by the culture method of the present invention are not particularly limited, and are, for example, mouse monoclonal antibodies, humanized monoclonal antibodies, or human monoclonal antibodies. Further, the class of immunoglobulin is not particularly limited, but for example, it is IgG (eg, IgG1, IgG2, etc.).

In the culture method or production method of the present invention, when culturing, containers or devices commonly used for culture can be used. Examples include multiwell plates, petri dishes, culture flasks, spinner flasks, jar fermenters, fermenters, roller bottles, hollow fibers, microcarriers, and the like.

The culture conditions in this embodiment may be normal animal cell culture conditions, such as a 5% CO ₂ atmosphere and a temperature of 37°C.

To collect cells from a culture solution, in the case of floating cells, for example, the culture solution is directly collected using a centrifuge or filter. In the case of adherent cells, for example, a 0.25% trypsin-0.02% EDTA solution is added to suspend the cells, and then the cells are collected by centrifugation or filtration.

Antibodies produced by cell culture can be harvested from a supernatant obtained by filtration or centrifugation if the substance accumulates in the culture medium. In addition, in the case of substances that accumulate within cells, cells obtained by filtration or centrifugation are subjected to homogenization, ultrasonic treatment, chemical treatment, etc. to obtain a supernatant liquid from which products are extracted.

In order to separate and purify the antibody from the supernatant, known methods can be appropriately combined. For example, ammonium sulfate precipitation, dialysis, ultrafiltration, electrophoresis, gel filtration, ion exchange chromatography, reversed phase chromatography, affinity chromatography, etc. are applied.

<Protein stabilizer>
The protein stabilizer of the present invention has an amine oxide group and has a mass average molecular weight of 2.
,000 or more.
The vinyl polymer (A) of the present invention only needs to have an amine oxide group and a mass average molecular weight of 2,000 or more. Conventionally known polymers can be used, and two or more kinds can be used. It may be used in combination, and by having an amine oxide group, it exhibits an excellent protein stabilizing effect.

In the present invention, the vinyl polymer having amine oxide groups can be obtained by the following two methods.
That is, a vinyl polymer having an amine oxide group can be obtained by polymerizing a monomer having an amine oxide group and another monomer.
Alternatively, after obtaining a vinyl polymer having a tertiary amino group, which can be called a precursor functional group for an amine oxide group, the tertiary amino group is reacted with an oxidizing agent to introduce an amine oxide group into the vinyl polymer. The latter method is preferable because it allows for the formation of side reactions and is less likely to cause side reactions. Note that reacting a tertiary amino group with an oxidizing agent is also referred to as "oxidation" hereinafter.

Specifically, the vinyl polymer preferably contains at least one of the structures represented by the above-mentioned general formulas 1 to 3, and among them, those containing the structure represented by general formula 1 are particularly preferable. When the amine oxide group is represented by the following formula, it is possible to exhibit suitable water solubility, suppress excessive interaction with proteins, and appropriately control the surrounding environment.
The vinyl polymer is preferably a copolymer, and may be any of a block copolymer, random copolymer, and alternating copolymer.

Vinyl polymers having such a structure can be obtained in two ways, as described above. More preferred is a method in which the tertiary amino group-containing monomer (a) and other monomers are polymerized and then oxidized. Other monomers preferably include a monomer (c) having an alkyl group having 4 to 18 carbon atoms.

<Tertiary amino group-containing monomer (a)>
Among the tertiary amino group-containing monomers (a) as precursors before oxidation, those for forming the structure of general formula 1 include:
For example, N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N, N-dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylamino Vinyl propionate, N,N-diethylaminovinyl propionate, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylaminoethylstyrene, N,N- Dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinylamine, or an acid anhydride containing an unsaturated group such as maleic anhydride, itaconic anhydride, citraconic anhydride, and N,N-dimethyl- Examples include reaction products with 1,3-propanediamine and the like, and reaction products between epoxy group-containing unsaturated compounds such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propanediamine.
Preferably, it is a (meth)acrylate monomer, more preferably N,N-dialkylaminoethyl (meth)acrylate or N,N-dialkylaminopropyl (meth)acrylate.
In the present invention, "(meth)acrylic" refers to both "acrylic" and "methacrylic."

For forming the structure of general formula 3, for example,
2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine , 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, 2-(t-butyl)-5-vinylpyridine, and the like.

For forming the structure of general formula 2, for example,
Examples include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 4-(t-butyl)-1-vinylimidazole, and the like.

<Other monomers>
When obtaining a vinyl polymer, other monomers having one ethylenically unsaturated group in one molecule can be used in addition to the monomer (a). By introducing a structure based on other monomers, polarity and Tg can be appropriately controlled, and not only can it have excellent coating properties and durability of the coated film, but also solvent solubility and the like can be controlled.
As other monomers, it is preferable to use a monomer (c) having an alkyl group having 4 to 18 carbon atoms from the viewpoint of protein stabilizing effect.

[Monomer (c) having an alkyl group having 4 to 18 carbon atoms]
The monomer (c) having an alkyl group having 4 to 18 carbon atoms is preferably a monomer having one ethylenically unsaturated group and an alkyl group having 4 to 18 carbon atoms in one molecule. Since the vinyl polymer (A) has an alkyl group having 4 to 18 carbon atoms, the polarity can be controlled and the surrounding environment can be appropriately controlled.

The monomer (c) is not particularly limited, but for example,
Butyl (meth)acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate, Alkyl (meth)acrylates such as stearyl (meth)acrylate, cetyl (meth)acrylate, cyclohexyl (meth)acrylate;
Examples include α-olefin ethylenically unsaturated monomers such as 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene, and 1-decene.

[Monomers (d) other than (a) and (c)]
Monomers (d) other than (a) and (c) can be copolymerized with monomers (a) and (c), and have one ethylenically unsaturated group in one molecule other than monomers (a) and (c). It is preferable that the monomer has the following.

The monomer (d) is not particularly limited, but
Methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, etc. Alkyl (meth)acrylate, isobonyl (meth)acrylate, methoxyethyl (meth)acrylate, ethoxyethyl (meth)acrylate, propoxyethyl (meth)acrylate , acrylic ester (meth)acrylates such as butoxyethyl (meth)acrylate, ethoxypropyl (meth)acrylate;
Aromatic ester (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, phenoxyethyl (meth)acrylate;
Allyl (meth)acrylate, 1-methylallyl (meth)acrylate, 2-methylallyl (meth)acrylate, 1-butenyl (meth)acrylate, 2-butenyl (meth)acrylate, 3-(meth)acrylate Butenyl, 1,3-methyl-3-butenyl (meth)acrylate, 2-chlorallyl (meth)acrylate, 3-chlorallyl (meth)acrylate, o-allylphenyl (meth)acrylate, (meth)acrylic acid 2-(allyloxy)ethyl, allyllactyl (meth)acrylate, citronellyl (meth)acrylate, geranyl (meth)acrylate, rhodinyl (meth)acrylate, cinnamyl (meth)acrylate, diallyl maleate, diallylitaconic acid, Ethylenically unsaturated group-containing (meth)acrylic acid esters such as vinyl (meth)acrylate, vinyl crotonate, vinyl oleate, vinyl linolenate, and 2-(2'-vinyloxyethoxy)ethyl (meth)acrylate. ;
Perfluoromethylmethyl (meth)acrylate, perfluoroethylmethyl (meth)acrylate, 2-perfluorobutylethyl (meth)acrylate, 2-perfluorohexylethyl (meth)acrylate, 2-perfluorooctylethyl (meth)acrylate , 2-perfluoroisononylethyl (meth)acrylate, 2-perfluorononylethyl (meth)acrylate, 2-perfluorodecylethyl (meth)acrylate, perfluoropropylpropyl (meth)acrylate, perfluorooctylpropyl (meth)acrylate ) acrylate, perfluorooctyl amyl (meth)acrylate, perfluorooctyl undecyl (meth)acrylate, etc. (meth) such as perfluoroalkyl group-containing ethylenically unsaturated monomers having a perfluoroalkyl group having 1 to 20 carbon atoms. Acrylate monomer;
2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-(meth)acryloyloxyethyl-2-hydroxyethylphthalate, glycerol mono(meth)acrylate , 4-hydroxyvinylbenzene, 1-ethynyl-1-cyclohexanol, allyl alcohol, and other hydroxyl group-containing monomers;
Maleic acid, fumaric acid, itaconic acid, citraconic acid, or alkyl or alkenyl monoesters thereof, β-(meth)acryloxyethyl phthalate monoester, β-(meth)acryloxyethyl isophthalate monoester, terephthalic acid A monomer having a carboxylic acid group, such as β-(meth)acryloxyethyl monoester, β-(meth)acryloxyethyl succinate monoester, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, or its anhydride;
Monomers having sulfonic acid groups such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(meth)acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl-(meth)acrylamide, N-ethoxymethyl-(meth)acrylamide, N-propoxymethyl-(meth)acrylamide, N-butoxymethyl-(meth)acrylamide, ) acrylamide, N-pentoxymethyl-(meth)acrylamide, N,N-di(methoxymethyl)acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N,N-di(ethoxymethyl)acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N,N-di(propoxymethyl)acrylamide, N-butoxymethyl-N-(propoxymethyl)methacrylamide, N,N-di(butoxymethyl)acrylamide, N-butoxymethyl -N-(methoxymethyl)methacrylamide, N,N-di(pentoxymethyl)acrylamide, N-methoxymethyl-N-(pentoxymethyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide , monomers having primary to tertiary amide groups such as diacetone (meth)acrylamide;
(meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamido-1,1-dimethylpropyl) ammonium chloride, trimethyl-3-(1-(meth)acrylamidopropyl) ammonium chloride, and a monomer having a quaternary amino group such as trimethyl-3-(1-(meth)acrylamido-1,1-dimethylethyl)ammonium chloride;
Polyethylene glycol (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, propoxypolyethylene glycol (meth)acrylate, n-butoxypolyethylene glycol (meth)acrylate, n-pentoxypolyethylene glycol (meth)acrylate Acrylate, phenoxypolyethylene glycol (meth)acrylate, polypropylene glycol (meth)acrylate, methoxypolypropylene glycol (meth)acrylate, ethoxypolypropylene glycol (meth)acrylate, propoxypolypropylene glycol (meth)acrylate, n-butoxypolypropylene glycol (meth)acrylate , n-pentoxypolypropylene glycol (meth)acrylate, phenoxypolypropylene glycol (meth)acrylate, polytetramethylene glycol (meth)acrylate, methoxypolytetramethylene glycol (meth)acrylate, phenoxytetraethylene glycol (meth)acrylate, hexaethylene Monomers having polyether chains such as glycol (meth)acrylate and methoxyhexaethylene glycol (meth)acrylate;
(Meth)acrylate monomers with a polymer structure in the side chain, such as ethylenically unsaturated compounds with polyester chains such as lactone-modified (meth)acrylate; styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allyl Aromatic vinyl monomers such as benzene and ethynylbenzene; ethylenically unsaturated monomers containing nitrile groups such as (meth)acrylonitrile; vinyl acetate, vinyl butyrate, vinyl propionate, vinyl hexanoate, vinyl caprylate, vinyl laurate, palmitic acid Fatty acid vinyl compounds such as vinyl and vinyl stearate; vinyl ether type ethylenically unsaturated monomers such as butyl vinyl ether and ethyl vinyl ether; allyl monomers such as allyl acetate and allyl cyanide; vinyl cyanide, vinyl cyclohexane, vinyl methyl ketone, etc. Vinyl monomers; ethynyl monomers such as acetylene and ethynyltoluene; perfluoroalkyls such as perfluorobutylethylene, perfluorohexylethylene, perfluorooctylethylene, and perfluorodecylethylene; perfluoroalkyl group-containing ethylenically unsaturated compounds such as alkylenes; Examples include monomers having ethylenically unsaturated bonds that are not (meth)acrylates, such as.

<Copolymer composition of vinyl polymer (A)>
The copolymer composition of polymer (a) will be explained.
The tertiary amino group-containing monomer (a) is preferably contained in an amount of 20 to 99.9% by mass, more preferably 65 to 85% by mass, based on the total amount of all monomers. Within the above range, a particularly excellent protein stabilizing effect is exhibited. The monomer (c) having an alkyl group having 4 to 18 carbon atoms is preferably contained in an amount of 0.1 to 80% by mass, more preferably 12 to 40% by mass, based on the total amount of all monomers.
The other monomer (d) is preferably contained in an amount of 0 to 79.9% by mass, more preferably 0 to 50% by mass, particularly preferably 0 to 25% by mass, based on the total amount of all monomers.

<Oxidation>
The tertiary amine group derived from monomer (a) can be converted into an amine oxide group by using various oxidizing agents. Thereby, an amine oxide group can be introduced into the vinyl polymer (A). The oxidation may be performed either before or after the polymerization of the vinyl polymer (A), and the oxidation before the polymerization is performed by adding the tertiary amino group-containing monomer (a) to the solution containing the tertiary amino group-containing monomer (a). In this method, an oxidizing agent is added to a solution containing a vinyl polymer obtained by polymerizing a monomer containing a tertiary amino group-containing monomer (a), and the mixture is heated at a temperature of 20° C. to 100° C. for 0.1 to 100 hours, preferably. The tertiary amino group can be oxidized by reacting for 1 to 50 hours.

As the oxidizing agent, an oxidizing agent such as peroxide or ozone is used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, and t-butyl hydroperoxide. Hydrogen peroxide is preferred, and is usually used in an aqueous solution. used in the form Although there are differences in degree, peroxide also functions as a radical generator, so in the case of vinyl polymers that use the tertiary amino group-containing unsaturated monomer (A) as an essential raw material, Oxidation is preferably performed after polymerization.

Generally, the amount of the oxidizing agent used is 0.2 to 3 times the molar equivalent of the oxidizable functional group, that is, the tertiary amino group, and further 0.5 to 2 times the molar equivalent. More preferably, molar equivalents are used. The obtained polymer solution can also be used after treating residual peroxide by a known method. Specifically, reducing agent addition treatment, ion exchange treatment, activated carbon treatment, treatment using a metal catalyst, etc. can be mentioned. The obtained polymer solution can be used as it is, but if necessary, the amine oxide group-containing polymer can also be isolated and used by known methods such as reprecipitation and solvent distillation. Further, the isolated amine oxide group-containing polymer can be further purified by reprecipitation, solvent washing, membrane separation, adsorption treatment, etc., if necessary.

As mentioned above, the vinyl polymer in the present invention includes those obtained by oxidizing the tertiary amino group-containing unsaturated monomer (a) and then polymerizing it with other monomers, and the tertiary amino group-containing unsaturated monomer (a) In addition to oxidized monomers obtained by polymerizing monomers that require Copolymerization using a reaction product of a group-containing unsaturated compound and an amine oxide group-containing compound such as hydroxyethyl-N,N-dimethylamine oxide can also be used.

<Amine oxide group content>
Amine oxide groups have the effect of appropriately controlling the surrounding environment of proteins and suppressing functional deactivation. The amine oxide group content in the polymer (A) is preferably 1 to 10 mmol/g, more preferably 2 to 8 mmol/g. By having the content of amine oxide groups within the above range, it is possible to exhibit suitable water solubility, suppress excessive interaction with proteins, and appropriately control the surrounding environment.

When obtaining the polymer (A) by polymerizing monomers having amine oxide groups, the content of amine oxide groups in the vinyl polymer (A) can be determined from the amount of monomers having amine oxide groups used in the polymerization. I can do it. On the other hand, in the case of oxidizing a polymer obtained after polymerizing a monomer that essentially includes the tertiary amino group-containing monomer (a), it can be calculated using the above-mentioned formula 1.

<Mass average molecular weight (Mw)>
The weight average molecular weight of the vinyl polymer (A) is preferably 2,000 to 10,000,000, more preferably 5,000 or more, still more preferably 10,000 or more, and particularly preferably 20,000 or more. ,000 or more. More preferably, it is 500,000 or less, particularly preferably 200,000 or less. When the molecular weight is within the above range, the thickening effect can be reduced and problems such as gelation can be prevented. Furthermore, handling such as pipetting becomes easier.

The mass average molecular weight of the vinyl polymer (A) was determined by gel permeation chromatography (GPC) in terms of standard pullulan. The following Condition 1 was used as the measuring device and measurement conditions. For other matters, JISK7252-1 to 4:2008 was referred to. Note that poorly soluble polymer compounds were measured at soluble concentrations under the following conditions. In addition, when it was difficult to measure the molecular weight of the vinyl polymer (A), the weight average molecular weight of the amine oxide precursor polymer was taken as the weight average molecular weight of the vinyl polymer (A). The mass average molecular weight of the amine oxide precursor polymer was determined by gel permeation chromatography (GPC) in terms of standard polystyrene, and the measurement device and measurement conditions were based on Condition 2 below.

(Condition 1)
Column: OHpak SB-G,
3 OHpak SB-806M HQ and
OHpak SB-802.5 HQ connected.
Carrier: 1/15 mol/L pH 7.0 phosphate buffer (phosphate buffer powder 1/15 mol/L pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 1 L of ion-exchanged water. thing)
Carrier flow rate: 1.0mL/min
Sample concentration: 0.5% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL

(Condition 2)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<Protein stabilization method>
By allowing the protein stabilizer of the present invention to coexist with the protein, the protein can be stored stably. The stabilizer and protein can coexist by dissolving or dispersing them in a common solvent.

The solvent in which the stabilizer and protein are allowed to coexist is preferably an aqueous solvent. Examples include water, phosphate buffered saline, and Tris buffered saline. The water may partially contain an organic solvent that is miscible with water, such as methanol, ethanol, propyl alcohol, or tetrahydrofuran.

The stabilizer of the present invention is used in an amount of preferably 100 times, more preferably 10,000 times the weight of the protein. If the amount of stabilizer added is insufficient, the stabilizing effect may not be achieved. For the same reason, the stabilizer is preferably added in an amount of 0.01% or more, more preferably 0.1% or more, based on the total amount of the solution.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the following Examples do not limit the scope of the present invention in any way. In addition, "part" and "%" in Examples and Comparative Examples represent "part by mass" and "% by mass", mol represents the amount of substance, and mol% represents the proportion of the amount of substance in the total monomer. represent.

<Method for measuring mass average molecular weight (Mw)>
The mass average molecular weight of the vinyl polymer (A) was determined by gel permeation chromatography (GPC) in terms of standard polystyrene. The measuring device and measurement conditions were basically based on Condition 1 below, and Condition 2 was used depending on the solubility of the sample. However, depending on the polymer species, a more appropriate carrier (eluent) and column suitable for it were selected as appropriate. Other matters were based on JISK7252-1~4:2008. Note that poorly soluble polymer compounds were measured at soluble concentrations under the following conditions.
In addition, when it was difficult to measure the molecular weight of the polymer, the weight average molecular weight of the amine oxide precursor polymer was taken as the weight average molecular weight of the polymer. For the mass average molecular weight of the amine oxide precursor polymer, a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) was used, and the measurement device and measurement conditions were in accordance with Condition 3 below.
(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
A combination of TOSOHTSKgelSuperHZ4000 and TOSOHTSKgelSuperHZ2000.
Carrier: Tetrahydrofuran Measurement temperature: 40℃
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 2)
Column: Two TOSOHTSKgelSuperAWM-H columns connected.
Carrier: 10mM LiBr/N-methylpyrrolidone Measurement temperature: 40°C
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<<Example of blocking agent for biochemical analysis>>
<Production of blocking agent 1>
[Manufacture example 1]
(Preparation of vinyl polymer (A))
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen inlet tube, a reflux condenser, and a dropping tube, 150 parts by mass of ethyl acetate was charged as an organic solvent under a nitrogen stream, and the mixture was heated at 80° C. for 30 minutes with stirring. In the dropping tube, 80 parts by mass of N,N-dimethylaminopropylacrylamide as a monomer, 20 parts by mass of butyl acrylate, 0.5 parts by mass of azobisisobutyronitrile as a polymerization initiator, and 10 parts by mass of ethyl acetate as a solvent. It was prepared and dripped over 2 hours. After completion of the dropping, the mixture was aged for 6 hours. Thereafter, the reaction was stopped by cooling to room temperature.
Next, 150 parts of ethanol and 50 parts of 35% hydrogen peroxide as an oxidizing agent (equimolar amount to the N,N-dimethylaminopropyl methacrylate used) were added, and the amino groups were reacted at 70°C for 16 hours. Oxidation was performed. Thereafter, the solvent was removed using a diaphragm pump to obtain a vinyl polymer (A).
The amine oxide group content of the obtained polymer was 4.73 mmol/g based on the amount of N,N-dimethylaminopropyl methacrylate added and the above formula. In addition, 1 g of the obtained vinyl polymer (A) was put into 99 g of ion-exchanged water at 25° C., stirred and dissolved, and then left at 25° C. for 24 hours. As a result, these resins showed no separation or precipitation, indicating that they were completely soluble and water-soluble.

(Production of blocking agent)
The vinyl polymer (A) obtained above was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain a blocking agent of Production Example 1 having a concentration of 5% by mass.

[Production Examples 2 to 15]
Vinyl polymer (A) was synthesized using the formulation shown in Table 1 in the same manner as in Production Example 1, and dissolved in a PBS solution to obtain blocking agents of Production Examples 2 to 15. The vinyl polymer (A) in Production Example 15 was obtained by polymerizing monomers in the same manner as in Production Example 1, and removing the solvent without reacting the oxidizing agent.

The symbols in Table 1 are as follows.
DMAPAA: N,N-dimethylaminopropylacrylamide DEAEMA: N,N-diethylaminoethyl methacrylate VP: Vinylpyridine VI: Vinylimidazole BA: Butyl methacrylate 2EHA: 2-ethylhexyl acrylate ISTA: Isostearyl acrylate MMA: Methyl methacrylate St: Styrene AA : Acrylic acid HEA: Hydroxyethyl acrylate

<Processing with blocking agent and performance evaluation>
[Actin attachment]
Human platelet-derived actin (manufactured by Cytoskeleton) was diluted with a PBS solution to 1% by mass to prepare an actin solution for evaluation. Two nitrocellulose membranes (Membrane L-08002-010 manufactured by As One) were each dropped with 2 μL of the actin solution for evaluation into one location using a micropipettor, and left to stand to dry.

[Processing with blocking agent]
Next, the nitrocellulose membrane to which actin was attached was placed in the blocking agent prepared above, and the membrane was shaken at room temperature for 1 hour to perform a blocking treatment. Thereafter, the nitrocellulose membrane was taken out from the blocking agent, placed in a PBS solution, and shaken for 15 minutes at room temperature. The PBS solution was renewed and the same washing procedure was repeated once more to remove excess blocking agent.

[Attachment of two types of antibodies to actin]
Anti-β-actin, monoclonal antibody, peroxidase conjugate (manufactured by Wako Pure Chemical Industries, Ltd.)
It was dissolved in a PBS solution to obtain a diluted actin antibody solution with a concentration of 0.01% by mass. Similarly, anti-GAPDH, monoclonal antibody, peroxidase conjugate (manufactured by Wako Pure Chemical Industries, Ltd.) was
A diluted GAPDH antibody solution having a concentration of 0.01% by mass was obtained by dissolving in S solution. The aforementioned nitrocellulose membrane from which excess blocking agent had been removed was placed in each antibody dilution solution and shaken at room temperature for 1 hour. Next, the nitrocellulose membrane was taken out from the antibody dilution solution, placed in a PBS solution, and shaken at room temperature for one hour to remove excess antibody. Note that GAPDH is an abbreviation for glyceraldehyde-3-phosphate dehydrogenase.

[Staining and evaluation]
Dissolve 10 mg of DAB tablets (3,3'-diaminobenzidine tetrahydrochloride, Wako Pure Chemical Industries, Ltd.) in 50 mL of 0.05 mol/L Tris-HCl buffer, and add 10 μL of 30% hydrogen peroxide to make a staining solution. It was adjusted. The two nitrocellulose membranes from which excess antibodies had been removed were each covered with a staining solution, and the excess staining solution on the surface was removed with a towel. According to the following criteria, the staining state of the actin attachment site when two types of antibody dilutions were used was evaluated, and the evaluation results are shown in Table 2.

"When using actin antibody dilution"
○: Clear staining △: Almost no staining ×: No staining "When using GAPDH antibody diluted solution"
○: No staining △: Staining ×: Clear staining "Comprehensive evaluation"
○: Staining occurs only when the actin antibody diluted solution is used, and there is no staining when the GAPDH antibody diluted solution is used.
△: Staining is weak when actin antibody dilution is used, and the contrast is small when using GAPDH antibody dilution, or staining occurs when actin antibody dilution is used, but GAPDH antibody dilution is used. Clear staining can also be seen in some cases.
×: The degree of staining is the same when using the actin antibody dilution solution and when using the GAPDH antibody dilution solution (no blocking performance)

As shown in Table 2, by using the blocking agent for biochemical analysis of the present invention, biochemical analysis of actin and actin antibodies could be performed. This is because the polymer (A) contained in the blocking agent for biochemical analysis of the present invention properly adsorbs to proteins, thereby not inhibiting antigen-antibody reactions and suppressing non-specific adsorption reactions. It is believed that there is.

On the other hand, in Comparative Examples 1 to 3, which used polymers that did not have 1.0 to 10.0 mmol/g of amine oxide groups, staining was observed even when a diluted GAPDH antibody was used, and the diluted actin antibody No clear difference was seen when using . Therefore, it was not possible to obtain sufficient sensitivity for analysis. This is considered to be because the blocking agent caused strong non-specific adsorption to both the antigen and the antibody, and did not exhibit blocking performance. However, Comparative Example 1 is a reference example.

<Production of blocking agent 2>
<Production of vinyl copolymer containing amine oxide group>
[Example 2-1]
In a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, 25.8 parts of butanol and 80 parts (78.3 mol%) of dimethylaminoethyl methacrylate as the tertiary amino group-containing vinyl monomer (a2) were added. , 20.0 parts (21.7 mol %) of butyl methacrylate was charged as a vinyl monomer (a3) containing an alkyl group having 1 to 18 carbon atoms. Furthermore, 3.0 parts of trimethylolpropane tris (3-mercaptopropionate) as a thiol group-containing compound (x) was added thereto to give a total amount of 3% of all vinyl monomers, and the mixture was sufficiently After replacing the atmosphere with nitrogen, the internal temperature was raised to 90°C. Separately, a mixture of 0.1 part of dimethyl 2,2'-azobis(2-methylpropinate) and 42.8 parts of butanol was prepared, and this was added in 13 portions. The butanol solution of dimethyl 2,2'-azobis(2-methylpropinate) was added for the first time when the reaction vessel was stabilized at 90°C.
Thereafter, it was added to the reaction vessel every 30 minutes. When the total reaction time reached 8 hours, the solid content was measured and it was confirmed that the conversion rate exceeded 98%, and the reaction was stopped by cooling to room temperature.
Next, 49.7 parts of 35% hydrogen peroxide solution (equimolar amount to the dimethylaminoethyl methacrylate used) was added as an oxidizing agent, and the tertiary amino group was oxidized by reacting at 80°C for 50 hours. Ta. Thereafter, butanol was removed using a diaphragm pump to obtain an amine oxide group-containing vinyl polymer (A-1).
The amine oxide group content of the obtained amine oxide group-containing vinyl polymer (A-1) was 4.58 mmol/g based on the added amount of dimethylaminoethyl methacrylate and the above-mentioned formula 1.
1 g of the obtained amine oxide group-containing vinyl polymer (A-1) was placed in 99 g of ion-exchanged water at 25° C., stirred and dissolved, and then left at 25° C. for 24 hours. As a result, these resins showed no separation or precipitation, indicating that they were completely soluble and water-soluble.

[Examples 2-2 to 2-11]
Amine oxide group-containing vinyl polymers (A-2 to A-11) were synthesized in the same manner as in Example 2-1 except that the composition shown in Table 2-1 was used.

[Comparative example 2-1]
In a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, add 25.8 parts of butanol, 70.0 parts (54.2 mol%) of butyl methacrylate, and 30 parts (45.8 mol%) of acrylic acid. , prepared. Furthermore, 3.0 parts of trimethylolpropane tris (3-mercaptopropionate) was added thereto as a thiol group-containing compound (x) to make the amount 3% based on the total amount of all vinyl monomers, and After the substitution, the internal temperature was raised to 90°C.Separately, a mixture of 0.1 part of dimethyl 2,2'-azobis(2-methylpropinate) and 42.8 parts of butanol was prepared, and this was mixed. The butanol solution of dimethyl 2,2'-azobis(2-methylpropinate) was added in 13 portions.The first addition was made when the reaction vessel was stabilized at 90°C, and then added every 30 minutes. When the total reaction time was 8 hours, the solid content was measured and it was confirmed that the conversion rate exceeded 98%, and the reaction was stopped by cooling to room temperature.Then, butanol was added using a diaphragm pump. was removed to obtain a vinyl copolymer (HA-1).

[Comparative example 2-2]
In a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, 25.8 parts of butanol and 50 parts (47.5 mol%) of dimethylaminoethyl methacrylate as the tertiary amino group-containing vinyl monomer (a2) were added. , 50.0 parts (52.5 mol %) of butyl methacrylate was charged as a vinyl monomer (a3) containing an alkyl group having 1 to 18 carbon atoms. Furthermore, 3.0 parts of trimethylolpropane tris (3-mercaptopropionate) as a thiol group-containing compound (x) was added thereto to give a total amount of 3% of all vinyl monomers, and the mixture was sufficiently After replacing the atmosphere with nitrogen, the internal temperature was raised to 90°C. Separately, a mixture of 0.1 part of dimethyl 2,2'-azobis(2-methylpropinate) and 42.8 parts of butanol was prepared, and this was added in 13 portions. The butanol solution of dimethyl 2,2'-azobis(2-methylpropinate) was added for the first time when the reaction vessel was stabilized at 90°C.
Thereafter, it was added to the reaction vessel every 30 minutes. When the total reaction time reached 8 hours, the solid content was measured and it was confirmed that the conversion rate exceeded 98%, and the reaction was stopped by cooling to room temperature. Thereafter, butanol was removed using a diaphragm pump to obtain a vinyl copolymer (HA-2).

The abbreviations in Table 2-1 are shown below.
DMAEMA: Dimethylaminoethyl methacrylate DEAPAA: Dimethylaminopropylacrylamide VP: Vinylpyridine VI: Vinylimidazole MMA: Methyl methacrylate BMA: Butyl methacrylate 2EHA: 2-ethylhexyl acrylate LMA: Lauryl methacrylate St: Styrene AA: Acrylic acid HEA: Hydroxyethyl acrylate TMMP: Trimethylolpropane tris (3-mercaptopropionate)
PEMP: Pentaerythritol tetrakis (3-mercaptopropionate)
DPMP: (dipentaerythritol hexakis (3-mercaptopropionate)

<Preparation of blocking agent>
[Example 2-13]
(1 solution of blocking agent)
The obtained amine oxide group-containing vinyl polymer (A-1) was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain a solution of blocking agent 1 with a solid content of 5% by mass.

[Examples 2-14 to 2-24, Comparative Examples 2-3 to 2-4]
(Blocking agent 2-13 solution)
The amine oxide group-containing vinyl polymer (A-1) was added to the amine oxide group-containing vinyl polymers (A-2 to A-11) and comparative vinyl polymers (HA-1 to HA-2). Solutions of blocking agents 2 to 13 were obtained in the same manner as the blocking agent 1 solution except for the following changes.
The vinyl copolymer (HA-1) used was one in which all acrylic acid was neutralized with dimethylaminoethanol. Furthermore, the vinyl copolymer (HA-2) used was one in which all the amino groups derived from dimethylaminoethyl methacrylate were neutralized with acetic acid.

<Evaluation of blocking agent>
[Antigen-antibody reaction test] and [non-specific adsorption test] were performed on the obtained blocking agent solution as described below, and evaluation was made based on the difference in absorbance. The results are shown in Table 2-2.

[Antigen-antibody reaction test]
(Solid-phase antibody treatment of well plate)
The solid phase CRP antibody ab8279 (abcam) was adjusted to a concentration of 25 μg/ml using a PBS solution. 50 μl of this solid-phase CRP antibody solution was added to each PSt 96-well plate, left to stand at room temperature for 2 hours, and then the solution was removed by suction.

(Antigen adsorption treatment)
CRP antigen 8C72 (manufactured by Hytest) was adjusted to 2 μg/ml using a PBS solution. 50 μl of this CRP antigen solution was added to each 96-well plate treated with the solid-phase antibody described above, and after standing at 4° C. for 12 hours, the solution was removed by suction.

(blocking process)
50 μl of the obtained blocking agent solution was added to each of the aforementioned antigen-treated 96-well plates, left to stand at room temperature for 2 hours, and then the solution was removed by suction.

(Labeled antibody adsorption treatment)
HRP-labeled CRP antibody ab24462 (abcam) was adjusted to 0.08 μg/ml with a PBS solution. 50 μl each of this HRP-labeled CRP antibody solution was added to the aforementioned blocking-treated 96-well plate, allowed to stand at room temperature for 2 hours, and then the solution was removed by suction. Thereafter, 200 μl of PBS solution was added to each well and removed by suction, and this was repeated three times.

(color reaction)
A coloring solution was prepared using 10 ml of a coloring agent from a coloring kit for peroxidase manufactured by Sumitomo Bakelite Co., Ltd. and 0.1 ml of a substrate solution. 100 μl of this coloring solution was added to each of the 96-well plates that had been subjected to the labeled antibody adsorption treatment described above, and allowed to stand at room temperature for 1 hour. Next, 100 μl of a stop solution from a color development kit for peroxidase manufactured by Sumitomo Bakelite was added to each well.

(Absorbance A measurement)
The absorbance at 450 nm (referred to as absorbance A) of each well was measured. This is the absorbance representing the amount of antibody adsorbed to the well due to antigen-antibody reaction (specific adsorption).

[Non-specific adsorption test]
(Solid-phase antibody treatment of well plate)
The solid phase CRP antibody ab8279 (abcam) was adjusted to a concentration of 25 μg/ml using a PBS solution. 50 μl of this solid-phase CRP antibody solution was added to a 96-well plate for ELISA manufactured by PSt, and after standing at room temperature for 2 hours, the solution was removed by suction.

(blocking process)
50 μl of the obtained blocking agent solution was added to each of the 96-well plates treated with the solid-phase antibody described above, and after standing at room temperature for 2 hours, the solution was removed by suction.

(Labeled antibody adsorption treatment)
HRP-labeled CRP antibody ab24462 (abcam) was adjusted to 0.08 μg/ml with a PBS solution. 50 μl each of this HRP-labeled CRP antibody solution was added to the aforementioned blocking-treated 96-well plate, allowed to stand at room temperature for 2 hours, and then the solution was removed by suction. Thereafter, 200 μl of PBS solution was added to each well and removed by suction, and this was repeated three times.

(color reaction)
A coloring solution was prepared using 10 ml of a coloring agent from a coloring kit for peroxidase manufactured by Sumitomo Bakelite Co., Ltd. and 0.1 ml of a substrate solution. 100 μl of this coloring solution was added to each of the 96-well plates that had been subjected to the labeled antibody adsorption treatment described above, and allowed to stand at room temperature for 1 hour. Next, 100 μl of a stop solution from a color development kit for peroxidase manufactured by Sumitomo Bakelite was added to each well.

(Absorbance B measurement)
The absorbance of each well at 450 nm (referred to as absorbance B) was measured. This is an absorbance that represents the amount of antibody adsorbed to the well due to non-specific adsorption of the labeled antibody since there is no antigen.

[Evaluation criteria]
◎: 1.0≦(Absorbance A-Absorbance B): Good 〇: 0.8≦(Absorbance A-Absorbance B)<1.0: Usable △: 0.5≦(Absorbance A-Absorbance B)<0 .8: Unusable ×: (Absorbance A - Absorbance B) <0.5: Defective

As shown in Table 2-2, by using the blocking agent for biochemical analysis of the present invention, biochemical analysis using CRP antigen-antibody reaction could be performed. Specifically, the blocking agents for biochemical analysis (Examples 2-13 to 2-24) using the amine oxide group-containing vinyl polymers (Examples 2-1 to 2-12) of the present invention are CRP There was a large difference in absorbance between the antigen-antibody reaction and the non-specific adsorption reaction, and the CRP antigen-antibody reaction could be detected with high sensitivity. That is, this is because the amine oxide group-containing vinyl polymer (A) contained in the blocking agent for biochemical analysis of the present invention has an amine oxide structure and a sulfur atom derived from a thiol group has been introduced into the copolymer. This is thought to be because the interaction with the antibody was appropriately controlled, the antigen-antibody reaction of CRP was not inhibited, and non-specific adsorption reactions other than the CRP antigen-antibody reaction could be suppressed. In particular, Examples 2-13 to 2-16 and 2-18 to 2-24, in which a branched structure was introduced into the copolymer by using a trifunctional or higher-functional thiol group-containing compound as a chain transfer agent, showed better results. Met.

On the other hand, a blocking agent for biochemical analysis (Comparative Example 2-3 to 2-4), the difference in absorbance between the CRP antigen-antibody reaction and the non-specific adsorption reaction was small, and it was not possible to obtain sufficient sensitivity for analysis. This means that if it does not have an amine oxide structure and a sulfur atom derived from a thiol group, it will not be possible to suppress nonspecific adsorption reactions other than the CRP antigen-antibody reaction, or it will inhibit the CRP antigen-antibody reaction itself. It is presumed that this is because.

<<Example of coating agent for inhibiting biofilm formation>>
<Method for measuring acid value>
The acid value is the mmol of potassium hydroxide required to neutralize the acid groups contained in 1 g of resin, and the measurement method may be any known method, and is generally carried out according to JIS K0070. The method is shown below. Accurately weigh 0.5 to 2 g of the sample (solid content: Sg). Add 10 mL of neutral ethanol to the accurately weighed sample to dissolve it. The obtained solution is subjected to potentiometric titration with a 0.1 mol/l ethanolic potassium hydroxide solution (potency: F). The point at which the potential difference curve reached a maximum was defined as the end point, and the titration amount (AmL) at this time was used to determine the acid value according to the following (Equation 2).
(Formula 2) Acid value (mmol/g) = (A x F x 0.1)/S

<Method for measuring amine value>
The amine value is mmol of potassium hydroxide, which is equivalent to the equivalent of hydrochloric acid required to neutralize the amino groups contained in 1 g of resin. The amine value was measured by the following method. Accurately weigh 0.5 to 2 g of the sample (solid content: Sg). Add 10 mL of neutral ethanol to the accurately weighed sample to dissolve it. The obtained solution is subjected to potentiometric titration with a 0.1 mol/l ethanolic hydrochloric acid solution (potency: f). The point at which the potential difference curve reached a maximum was defined as the end point, and the amine value was determined using the titration amount (AmL) at this time according to the following (Equation 3).
(Formula 3) Amine value (mmol/g) = (A x f x 0.1)/S

<Synthesis of amine oxide group-containing polymer>
(Polymer solution (P-1))
100 parts of ethyl acetate was charged into a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, and the internal temperature was raised to 75° C. and the mixture was sufficiently purged with nitrogen. Separately prepared 0.3 parts of 2,2'-azodiisobutyronitrile, 20 parts of N,N-dimethylaminoethyl methacrylate as the monomer (A), 5 parts of acrylic acid as the monomer (B), A mixture of 75 parts of butyl acrylate as monomer (C) was added dropwise for 3 hours while maintaining the internal temperature at 75°C, and stirring was continued for an additional 5 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, the solution was cooled to obtain a solution of a polymer having a tertiary amino group.
Next, to the obtained solution of the polymer having a tertiary amino group, 14.4 parts of 30% hydrogen peroxide was added as an oxidizing agent (equimolar amount to the N,N-dimethylaminoethyl methacrylate used), Amino groups were oxidized by reacting at 70°C for 16 hours. After confirming that the amine oxide conversion rate exceeded 99%, it was cooled and taken out, the solvent was completely volatilized in an oven, and then dissolved in ethanol to obtain a polymer solution (P-1) with a solid content of 50% by mass. I got it. The above amine oxide conversion rate was determined according to the method disclosed in JP-A-10-182589.
The amine oxide group content of the obtained polymer was 1.25 mmol/g based on the amount of N,N-dimethylaminoethyl methacrylate added and the above-mentioned formula 1. Furthermore, the mass average molecular weight of the obtained polymer was measured by GPC and found to be 60,000.

(Polymer solution (P-2 to 18, 20))
Synthesis was carried out in the same manner as the polymer solution (P-1) according to the composition and charged mass parts in Table 3-1 to obtain polymer solutions (P-2 to P-18, 20).

(Polymer solution (P-19))
100 parts of ethyl acetate was charged into a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, and the internal temperature was raised to 75° C. and the mixture was sufficiently purged with nitrogen. A mixture of 0.3 parts of 2,2'-azodiisobutyronitrile, 5 parts of acrylic acid as monomer (B), and 95 parts of butyl acrylate as monomer (C), which had been prepared separately, was mixed at an internal temperature. The dropwise addition was continued for 3 hours while maintaining the temperature at 75°C, and stirring was continued for an additional 5 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, it was cooled and taken out, and diluted with ethyl acetate to obtain a polymer solution (P-19) with a solid content of 50% by mass.

(Polymer solution (P-21))
100 parts of ethyl acetate was charged into a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, and the internal temperature was raised to 75° C. and the mixture was sufficiently purged with nitrogen. Separately prepared 0.3 parts of 2,2'-azodiisobutyronitrile, 20 parts of N,N-dimethylaminoethyl methacrylate as the monomer (A), 5 parts of acrylic acid as the monomer (B), A mixture of 95 parts of butyl acrylate as monomer (C) was added dropwise for 3 hours while keeping the internal temperature at 75°C, and stirring was continued for an additional 5 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, it was cooled and taken out, and diluted with ethanol to obtain a polymer solution (P-21) having a tertiary amino group with a solid content of 50% by mass.

(Polymer solution (P-22))
60 parts of ethyl acetate and 40 parts of ion-exchanged water were charged into a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, and the internal temperature was raised to 75° C. and the mixture was sufficiently purged with nitrogen. Separately prepared 0.3 parts of 2,2'-azodiisobutyronitrile, 5 parts of acrylic acid as monomer (B), 75 parts of butyl acrylate as monomer (C), and 20 parts of methacroylcholine chloride. The mixture was added dropwise for 3 hours while maintaining the internal temperature at 75°C, and stirring was continued for an additional 5 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, it was cooled, taken out, and diluted with ethanol to obtain a polymer solution (P-22) having a quaternary amino group with a solid content of 50% by mass.

The characteristic values of the obtained polymer are shown in Table 3-1.

The abbreviations in Table 3-1 are shown below.
DMAEMA: N,N-dimethylaminoethyl methacrylate DEAEMA: N,N-diethylaminoethyl methacrylate DMAPAA: N,N-dimethylaminopropylacrylamide VP: 4-vinylpyridine VI: 2-methyl-1-vinylimidazole AA: Acrylic acid HEA :2-hydroxyethyl acrylate GMA: glycidyl methacrylate BA: butyl acrylate OA: n-octyl arylate MMA: methyl methacrylate TMAEMC: methacroylcholine chloride AIBN: 2,2'-azodiisobutyronitrile

<Adjustment of coating agent for inhibiting biofilm formation>
[Example 3-1]
(Biofilm formation inhibiting coating agent 1)
4.0 parts of the obtained amine oxide group-containing polymer solution (P-1) and 2.0 parts of V-02 (manufactured by Nisshinbo Co., Ltd.; Carbodilite V-02) as a crosslinking agent were mixed, and 16.0 parts of ethanol was mixed. A coating solution for inhibiting biofilm formation 1 was obtained by diluting the solution with 0 parts to prepare a 10% coating solution.

[Examples 3-2 to 3-15, 3-17 to 3-26]
(Biofilm formation inhibiting coating agent 2-15, 17-26)
In the same manner as in Example 1, biofilm formation inhibiting coating agents 2 to 15 and 17 to 26 were prepared using the compositions and parts by mass shown in Table 3-2.

[Example 3-16]
(Biofilm formation inhibiting coating agent 16)
4.0 parts of the obtained amine oxide group-containing polymer (P-12) was diluted with 16 parts of ethanol to prepare a 10% coating solution to obtain biofilm formation inhibiting coating agent 16.

The abbreviations in Table 3-2 are shown below.
V-02: Carbodiimide group-containing compound (manufactured by Nisshinbo Co., Ltd.; Carbodilite V-02)
V-10: Carbodiimide group-containing compound (manufactured by Nisshinbo Co., Ltd.; Carbodilite V-10)
V-09: Isocyanate group-containing compound (manufactured by Nisshinbo Co., Ltd.; Carbodilite V-09)
MDI: Isocyanate-containing compound (2,2'-diphenylmethane diisocyanate)
40E: Epoxy group-containing compound (manufactured by Kyoeisha Chemical Co., Ltd.; Epolite 40E)
LV11: Amino group-containing compound (manufactured by Mitsubishi Chemical Corporation; epoxy resin curing agent jER Cure)
EtOH: Ethanol EtOAc: Ethyl acetate

<Evaluation of coating agent for inhibiting biofilm formation>
The biofilm formation inhibiting coating agents (coating liquids) obtained in Examples and Comparative Examples were evaluated as follows. The results are shown in Table 3-3.

[water resistance]
2.0 g of the obtained coating agent was added to a precisely weighed shallow metal container, and the container was heated and dried at 150° C. for 10 minutes. After taking it out of the oven and precisely weighing the shallow metal container, 5.0 g of ion-exchanged water was added to the shallow metal container and left overnight. After the ion-exchanged water was suctioned and discharged from the shallow metal container, it was dried again at 150° C. for 10 minutes, and the shallow metal container was accurately weighed. The solubility in water was calculated using the following formula, and the water resistance was evaluated based on a four-level evaluation standard.
Solubility in water (%) = 100-[(z-x)/(y-x)] x 100
x: Mass of shallow metal container (g)
y: Mass (g) before treatment with ion-exchanged water
z: Mass after treatment with ion-exchanged water (g)
◎: Solubility in water≦2%: Very good ○:2%<Solubility in water≦4%: Good △:4%<Solubility in water≦10%: Usable ×:10%<Solubility in water Solubility: Not usable

<Manufacture and evaluation of biofilm formation suppressing laminate>
The obtained coating agent 1-26 was placed on a 75 μm thick polyethylene terephthalate (PET) film (manufactured by Panac Co., Ltd.; Lumirror #75, surface ozone treated) with a bar so that the film thickness after drying was 1.0 μm. It was coated with a coater, dried at 80°C for 2 minutes, and then heated at 80°C for 24 hours to obtain laminate 1-26. Separately, Staphylococcus aureus (ATCC 25923) was precultured at 37° C. for 24 hours and allowed to grow. A 1% bacterial solution was prepared by adding the bacterial solution to phosphate buffered water (PBW).
Cut the obtained laminate into a size of 1.5 cm x 1.5 cm, set one in each well of a 24-well microplate (manufactured by Falcon) with the coated side facing upward, and add 1.5 cm of sterile water. 0 mL was added and immersed at 37°C for 24 hours.
Next, 1.0 mL of sterile water was removed from the 24-well microplate, 1.0 mL of a separately prepared Staphylococcus aureus solution was added, and cultured at 25° C. for 24 hours or at 25° C. for 168 hours, respectively. After culturing for 24 or 168 hours, the bacterial solution was removed, the coating was washed three times with 1.2 mL of sterile water, and 0.1% crystal violet aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added for 20 minutes. The biofilm was stained after being allowed to stand. Thereafter, it was washed three times with 1.2 mL of sterile water and air-dried to obtain a laminate with stained biofilm.
Crystal violet was extracted from the dyed laminate using 2.0 mL of a 33% acetic acid solution, and the absorbance of the extract was measured using a microplate reader.
Biofilm formation inhibitory properties were evaluated based on absorbance using the following four-level evaluation criteria. The results are shown in Table 3-3.
◎: Absorbance≦0.10: Very good ○: 0.10<Absorbance≦0.13: Good △:0.13<Absorbance≦0.20: Usable ×: 0.20<Absorbance: Unusable

From Table 3-3, since the polymer used in Comparative Example 1 did not have an amine oxide group, it had poor biofilm formation inhibiting properties after 24 hours and 168 hours of culture. The polymer used in Comparative Example 3-2 had a low molecular weight and the coated polymer peeled off, resulting in poor biofilm formation inhibitory properties after 24 hours of culture. The polymer used in Comparative Example 3-3 had a tertiary amino group but did not contain amine oxide, and therefore did not exhibit biofilm formation inhibiting properties. The polymer used in Comparative Example 3-4 had a quaternary amino group but did not contain amine oxide, and therefore did not exhibit biofilm formation inhibiting properties.
On the other hand, a biofilm formation inhibiting coating agent containing a polymer containing an amine oxide group and having a mass average molecular weight of 2,000 to 10,000,000 has excellent water resistance and long-term inhibition of biofilm formation. It was confirmed that it exhibited excellent performance effects. In particular, it was confirmed that when the amine oxide group content in the polymer (a) was 0.25 to 5 mmol/g, the optimal ability to inhibit biofilm formation was maintained even when immersed in water for a long time. Among them, when the concentration was 0.5 to 2 mmol/g, an extremely excellent long-term ability to inhibit biofilm formation was exhibited. Furthermore, it was shown that when a carboxyl group-containing monomer was used as the monomer (B) having a crosslinkable group, the water resistance and long-term biofilm formation suppressing ability were excellent.

<<Example of cell culture equipment treatment agent>>
<Method for measuring mass average molecular weight (Mw)>
The mass average molecular weight of the polymer (a) was determined by gel permeation chromatography (GPC) in terms of standard polystyrene. The measuring device and measurement conditions were basically based on Condition 1 below, and Condition 2 was used depending on the solubility of the sample. However, depending on the polymer species, a more appropriate carrier (eluent) and column suitable for it were selected as appropriate. Other matters were based on JISK7252-1~4:2008. Note that poorly soluble polymer compounds were measured at soluble concentrations under the following conditions.
In addition, when it was difficult to measure the molecular weight of polymer (a), the weight average molecular weight of the amine oxide precursor polymer was taken as the weight average molecular weight of polymer (a). For the mass average molecular weight of the amine oxide precursor polymer, a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) was used, and the measurement device and measurement conditions were in accordance with Condition 3 below.
(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
A combination of TOSOHTSKgelSuperHZ4000 and TOSOHTSKgelSuperHZ2000.
Carrier: Tetrahydrofuran Measurement temperature: 40℃
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 2)
Column: Two TOSOHTSKgelSuperAWM-H columns connected.
Carrier: 10mM LiBr/N-methylpyrrolidone Measurement temperature: 40°C
Carrier flow rate: 1.0mL/min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection volume: 0.1mL
(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSOHTSKgelSuperAW3000 and TOSOHTSKgelSuperAW2500.
Carrier: Mixture of N,N-dimethylformamide (1L), triethylamine (3.04g), LiBr (0.87g) Measurement temperature: 40°C
Carrier flow rate: 0.6mL/min

<Synthesis of vinyl polymer containing amine oxide group>
(Polymer (P-1))
100 parts of ethyl acetate was charged into a reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux device, and the internal temperature was raised to 75° C. and the mixture was sufficiently purged with nitrogen. Separately prepared 1.5 parts of 2,2'-azodiisobutyronitrile, 50 parts of N,N-dimethylaminoethyl methacrylate as the tertiary amino group-containing monomer (A), and other monomers (C). A mixture of 45 parts of butyl acrylate as a monomer (B) and a mixture of 5 parts of acrylic acid as a crosslinkable group-containing monomer (B) were added dropwise for 3 hours while maintaining the internal temperature at 75°C, and stirring was continued for an additional 2 hours. . After confirming that the conversion rate exceeded 98% by solid content measurement, the solution was cooled to obtain a solution of a polymer having a tertiary amino group.
Next, 30.9 parts of 35% hydrogen peroxide (equimolar amount to the N,N-dimethylaminoethyl methacrylate used) was added as an oxidizing agent to the obtained solution of the polymer having a tertiary amino group. Amino groups were oxidized by reacting at 70°C for 16 hours. After confirming that the amine oxide conversion rate exceeded 98%, it was cooled and taken out, and then the solvent was completely volatilized using a diaphragm pump to obtain an amine oxide group-containing polymer (P-1).
The amine oxide group content of the obtained (P-1) was 3.0 mmol/g based on the amount of N,N-dimethylaminoethyl methacrylate added and the above-mentioned formula 1. Moreover, when the mass average molecular weight of the obtained polymer was measured by GPC, it was 131,300.

(Polymer (P-2 to 9, 11))
Polymers (P-2 to P-9, 11) were synthesized in the same manner as Polymer (P-1) according to the composition and charged mass parts shown in Table 1.

(Polymer (P-10))
98.0 parts of 1-butanol was charged into a reaction vessel equipped with a gas inlet tube, a thermometer, a condenser, and a stirrer, and the mixture was purged with nitrogen gas. The inside of the reaction vessel was heated to 110°C, and a mixture of 20 parts of butyl acrylate, 50 parts of styrene, 30 parts of acrylic acid, and 1 part of 2,2'-azobis(isobutyronitrile) was added dropwise over 2 hours. A polymerization reaction was performed. After the dropwise addition was completed, the reaction was further carried out at 110° C. for 3 hours, and after confirming that the conversion rate exceeded 98% by solid content measurement, the reaction was stopped by cooling to room temperature. Thereafter, 1-butanol was removed using a diaphragm pump to obtain a polymer (P-10). Moreover, the mass average molecular weight of the obtained polymer was 133,000.

The abbreviations in Table 4-1 are shown below.
DMAEMA: N,N-dimethylaminoethyl methacrylate DEAEMA: N,N-diethylaminoethyl methacrylate DMAPAA: Dimethylaminopropylacrylamide VP: 2-vinylpyridine VI: 1-vinylimidazole DCPA: Dicyclopentenyloxyethyl acrylate BA: Butyl acrylate St: Styrene MEA: 2-methoxyethyl acrylate AA: Acrylic acid HEA: 2-hydroxyethyl acrylate AIBN: 2,2'-azodiisobutyronitrile HPO: 35% hydrogen peroxide

<Manufacture of cell culture equipment treatment agent>
[Example 4-1]
(Cell culture equipment treatment agent (PC-1))
10.0 parts of the obtained polymer (P-1) and 5.0 parts of a carbodiimide group-containing compound (manufactured by Nisshinbo Holdings, Inc.; Carbodilite V-02) as a crosslinking agent were diluted with ethanol to form a 10% solution. A cell culture equipment treatment agent (PC-1) was obtained.

[Examples 4-2 to 4-9, Comparative Examples 4-1 to 4-2]
(Cell culture equipment treatment agent (PC-2 to 11))
Cell culture equipment treatment agents (PC-2 to 11) were prepared in the same manner as (PC-1) except that the composition and parts by mass of the preparations were changed as shown in Table 2.

<Evaluation of cell culture equipment treatment agents>
The following evaluations were performed on the obtained cell culture equipment treatment agents (PC-1 to PC-11). The results are shown in Table 2.

[Evaluation item 1. <Water resistance>]
2.0 g of the cell culture equipment treatment agents (PC-1 to PC-11) were added to a precisely weighed shallow metal container and heated at 150° C. for 10 minutes to dry. After taking it out of the oven and precisely weighing the shallow metal container, 5.0 g of ion-exchanged water was added to the shallow metal container and left overnight. After the ion-exchanged water was suctioned and discharged from the shallow metal container, it was dried again at 150° C. for 10 minutes, and the shallow metal container was precisely weighed. The solubility in water was calculated using the following formula, and the water resistance was evaluated based on a four-level evaluation standard. The higher the water resistance, the better the durability.
Solubility in water (%) = 100-[(z-x)/(y-x)] x 100
x: Mass of shallow metal container (g)
y: Mass (g) before treatment with ion-exchanged water
z: Mass after treatment with ion-exchanged water (g)
(Evaluation criteria)
◎: Solubility in water≦2%: Very good○: 2%<Solubility in water≦4%: Good△:4%<Solubility in water≦10%: Usable ×:10%<Solubility in water Solubility: Not usable

The abbreviations in Table 4-2 are shown below.
V-02: Carbodiimide group-containing compound (manufactured by Nisshinbo Holdings, Inc.; Carbodilite V-02)
V-10: Carbodiimide group-containing compound (manufactured by Nisshinbo Holdings, Inc.; Carbodilite V-10)
MDI: Isocyanate-containing compound (2,2'-diphenylmethane diisocyanate)

<Manufacture and evaluation of cell culture equipment>
Using the obtained cell culture equipment treatment agent, cell culture equipment was produced as follows. Furthermore, the following evaluations were performed on the obtained cell culture equipment. The results are shown in Tables 4-3 to 4-5.

[Examples 4-10 to 4-18, Comparative Examples 4-3 and 4-4]
(Cell culture equipment (D-1 to 11))
Approximately 0.5 ml of cell culture equipment treatment agents (PC-1 to PC-11) were injected into each well of a U-shaped 96-well plate. After suctioning and discharging this, it was dried at 50°C for 3 hours to prepare cell culture equipment (D-1 to 11) with the inside of the well plate coated with the cell culture equipment treatment agent (PC-1 to 11). did.

[Evaluation item 2. <Spheroid formation test>]
After sterilizing the cell culture equipment (D-1 to D-11) with ethylene oxide gas, use Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as a medium, and use a mouse fibroblast cell line ( NIH/3T3 cells) were seeded at 1 x ¹⁰ cells per well and cultured in a 5% CO ₂ /37°C incubator until the 5th day, and the cell culture state after 5 days was photographed using a transmission light microscope at 40x magnification. and evaluated by observing cell morphology.
(Evaluation criteria)
○: One spheroid is formed △: Multiple spheroids are formed ×: No spheroid is formed

[Examples 4-19 to 4-27, Comparative Examples 4-5 and 4-6]
(Cell culture equipment (D-12-22))
Spin coat the cell culture equipment treatment agents (PC-1 to PC-11) onto the slide glass to a film thickness of 1.0 μm, heat treat at 90°C for 2 minutes, and apply the cell culture equipment treatment agents (PC-1 to PC-11) onto the glass. Cell culture equipment (D-12 to D-22) coated with PC-1 to PC-11) was obtained.

[Evaluation item 3. <Contact angle with water>]
The contact angle with water of the cell culture equipment treatment agent-coated surface of the obtained cell culture equipment (D-12 to D-22) was measured based on JISK 6788 (ISO8296).

[Evaluation item 4. <Antibody protein adsorption property>]
Cut the cell culture equipment (D-1 to 11) into 11 mm squares and place them in a U-shaped 24-well plate, and add HPR-IgG solution 1 diluted 10,000 times with phosphate buffered saline (PBS).
ml was added and soaked. After incubation for 1 hour at room temperature, PBS-T (0.1%
Each well was washed four times using Tween 20). After dispensing 1 ml of the staining solution to each well and incubating for 10 minutes at room temperature, dispensing 1 ml of the reaction stop solution to each well, measure the absorbance Aλ at 450 nm (subwavelength 650 nm) using a MITHRAS2 LD943-M2M microplate. Measured using a reader. The smaller the absorbance Aλ, the lower the antibody protein adsorption property and the better. Evaluation was made based on the following criteria.
Staining solution: TMBZ solution (3,3',5,5'-tetramethylbenzidine)
Reaction stop solution: Takara Bio Inc. WASH and Stop Solution For ELISA With Solution for ELISA without Sulfuric Acid
HPR-IgG: Enzyme, antibody (HORSERADIH PEROXIDASE IMMU
NOGLOBULING)
PBS-T: PBS with 0.1% Tween20 added (evaluation criteria)
◎: Aλ≦0.2 (extremely good)
○: 0.2<Aλ≦0.6 (good)
△: 0.6<Aλ≦0.8 (usable)
×: 0.8<Aλ (defective)

[Examples 4-28 to 4-36, Comparative Examples 4-7 and 4-8]
(Cell culture equipment (D-23-33))
Cell culture equipment treatment agents (PC-1 to 11) were poured into a PDMS (polydimethylsiloxane) standard chip (manufactured by Fluidware Technologies) channel having a channel width of 200 μm and height of 50 μm, and heated at 50°C. After leaving it in this state for 4 hours, it was washed three times with pure water and once with PBS (phosphate buffered saline, manufactured by Hayashi Pure Chemical Industries, Ltd.) to remove unadsorbed components, and the cell culture equipment ( D-23 to D-33) were obtained.

[Evaluation item 5. <Blood feeding test>]
In the obtained cell culture equipment (D-23 to 33), a blood sample was pumped under constant pressure at an inlet speed of 2 μL/min for 12 minutes, and 1 minute after the liquid was pumped, the blood sample was pumped from the flow path outlet for 1 minute. The amount of blood sample that flowed out, the amount of blood sample that flowed out from the flow path outlet in one minute from 5 minutes to 6 minutes after the start of liquid feeding, and the amount of blood sample that flowed out from the flow path outlet in 1 minute from 10 minutes to 11 minutes after the start of liquid feeding. The amount of blood sample that flowed out was measured. The larger the amount of blood sample that flows out and the more constant it is without decreasing over time, the better.

As shown in Tables 4-2 to 4-5, the cell culture equipment treatment agent of the present invention has high water resistance, and the cell culture equipment using the treatment agent has suppressed antibody protein adsorption properties and has excellent It showed blood-transferability. In particular, when the vinyl polymer had a crosslinkable group and contained a crosslinking agent, it had excellent water resistance. On the other hand, when polymer (a) having a number average molecular weight of less than 2,000 was used, the water resistance was significantly inferior. In addition, particularly when the amine oxide group content was 0.5 to 5 mmol/g, protein adsorption suppression and blood delivery performance were excellent.
As described above, the cell culture equipment treatment agent of the present invention has excellent water resistance, and the cell culture equipment made using the treatment agent has high spheroid formation properties, exhibits excellent protein and cell adhesion prevention effects, and has excellent blood flow resistance. It has also been shown to be useful as a medical device due to its excellent liquid properties.

<<Examples of cell culture medium additives>>
<Manufacture of cell culture medium additives>
[Example 5-1]
(Cell culture medium additive 1)
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen inlet tube, a reflux condenser, and a dropping tube, 150 parts by mass of ethyl acetate was charged as an organic solvent under a nitrogen stream, and the mixture was heated at 80° C. for 30 minutes with stirring. A dropping tube was charged with 80 parts by mass of dimethylaminopropylacrylamide as monomers, 20 parts by mass of butyl acrylate, 0.5 parts by mass of azobisisobutyronitrile as a polymerization initiator, and 10 parts by mass of ethyl acetate as a solvent, and left for 2 hours. It dripped. Heating was continued for 6 hours after the completion of the dropwise addition. Thereafter, the reaction was stopped by cooling to room temperature.
Next, 150 parts of ethanol and 50 parts of 35% hydrogen peroxide as an oxidizing agent (equimolar amount to the dimethylaminopropylacrylamide used) were added and reacted at 70°C for 16 hours to oxidize the tertiary amino group. I did it. Thereafter, the solvent was removed using a diaphragm pump to obtain a vinyl polymer (A-1) having amine oxide groups.
The amine oxide group content of the obtained vinyl polymer (A-1) was 4.73 mmol/g based on the amount of dimethylaminopropylacrylamide added and the above-mentioned formula 1.
Next, 1 g of the obtained vinyl polymer (A-1) was added to 99 g of phosphate buffered saline (hereinafter referred to as PBS) at 25°C, stirred and dissolved, and then heated at 25°C for 24 hours. Upon standing, cell culture medium additive 1 diluted to 1% by mass with PBS was obtained.
As a result, neither separation nor precipitation of the vinyl polymer (A-1) was observed, indicating that it was completely soluble and soluble in PBS.

[Examples 5-2 to 5-12, Comparative Examples 5-1 and 5-2]
(Cell culture medium additives 2 to 14)
Vinyl polymers with amine oxide groups (A-2 to A-12) and vinyl polymers without amine oxide groups were prepared in the same manner as Cell Culture Medium Additive 1, except that the composition was changed to the one shown in Table 5-1. After obtaining (A-13, 14), it was dissolved in PBS to obtain cell culture medium additives 2 to 14 diluted to 1% by mass with PBS.
Note that vinyl polymers (A-13, 14) that do not have amine oxide groups did not completely dissolve when diluted to 1% by mass with PBS after removing the solvent without reacting with the oxidizing agent. Although it was shown to be insoluble in PBS, the supernatant portion was used for evaluation.

The abbreviations in Table 5-1 are shown below.
DMAPAA: Dimethylaminopropylacrylamide DEAEMA: Diethylaminoethyl methacrylate VP: Vinylpyridine VI: Vinylimidazole BA: Butyl methacrylate 2EHA: 2-Ethylhexyl acrylate ISTA: Isostearyl acrylate MMA: Methyl methacrylate St: Styrene AA: Acrylic acid HEA: Hydroxyethyl acrylate

<Evaluation of cell culture medium additives>
The obtained cell culture medium additive was evaluated as follows. The results are shown in Table 5-2.

[Formation of cell aggregates]
The medium used was Dulbecco's modified Eagle medium (DMEM) to which 10% fetal bovine serum (FBS) was added (hereinafter also referred to as "10% FBS-DMEM medium"). A mouse fibroblast cell line (NIH/3T3 cells) was added to a 10% FBS-DMEM medium to prepare a cell suspension with an NIH/3T3 concentration of 100,000 cells/ml. 100 μL of the obtained cell suspension was seeded into each well of a sterilized polystyrene U-bottom 96-well plate. Furthermore, the obtained cell culture medium additives were added and pipetted so that the concentrations of the vinyl polymer (A) were 0.05% by mass and 0.5% by mass, respectively. Thereafter, the cells were cultured in a 5% CO ₂ /37°C incubator until the 5th day. The ability to form cell aggregates was evaluated by photographing the cell culture state on the 5th day using a transmission optical microscope at a magnification of 40 times and visually observing the morphology of the cells.
a: One spheroid is formed: Good b: Multiple spheroids are formed: Unusable c: No spheroid is formed: Poor

[Cellular ATP assay]
ATP (adenosine-5'-triphosphate) assay was performed on the cells cultured up to the 5th day, and the percentage of viable cells was evaluated. Specifically, 100 μl of ATP reagent (“clump” ATP measurement reagent: manufactured by Toyo B-Net Co., Ltd.) was added to the well after culture, pipetted 5 times, left to stand at room temperature for 5 minutes, and then 100 ml of the reagent was added. - The cell lysate was dispensed onto a separate plate and stirred for 1 minute. The amount of luminescence was measured using Mithras LB940 (manufactured by Berthold).
Proportion of living cells = (Luminescence amount after 5 days of culture) / (Luminescence amount when cultured for 5 days without adding cell culture medium additives) x 100
a: 50% or more: Good b: 20% or more but less than 50%: Usable c: Less than 20%: Poor

As shown in Table 5-2, by using the cell culture medium additive of the present invention, one spheroid was formed, indicating that cell aggregate formation was good. Furthermore, the results of the ATP assay showed that no cell death occurred and the number of viable cells was at a usable level. This is because the vinyl polymer (A) contained in the cell culture medium additive of the present invention properly adsorbs proteins, giving priority to adhesion between cells and inhibiting adhesion between cells and the substrate. It is believed that there is. On the other hand, in Comparative Examples 5-1 and 5-2 in which a vinyl polymer having no amine oxide group was used, no cell aggregates were formed. Furthermore, the results of the ATP assay showed that the percentage of viable cells had decreased significantly. This is considered to be because the environment around the cells could not be controlled appropriately due to the absence of amine oxide groups.

<Production of antibody-producing cell culture medium additive>
[Example 6-1]
(Cell culture medium additive 1)
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen inlet tube, a reflux condenser, and a dropping tube, 150 parts by mass of ethyl acetate was charged as an organic solvent under a nitrogen stream, and the mixture was heated at 80° C. for 30 minutes with stirring. A dropping tube was charged with 80 parts by mass of dimethylaminopropylacrylamide as monomers, 20 parts by mass of butyl acrylate, 0.5 parts by mass of azobisisobutyronitrile as a polymerization initiator, and 10 parts by mass of ethyl acetate as a solvent, and left for 2 hours. It dripped. Heating was continued for 6 hours after the completion of the dropwise addition. Thereafter, the reaction was stopped by cooling to room temperature.
Next, 150 parts of ethanol and 50 parts of 35% hydrogen peroxide as an oxidizing agent (equimolar amount to the dimethylaminopropylacrylamide used) were added and reacted at 70°C for 16 hours to oxidize the tertiary amino group. I did it. Thereafter, the solvent was removed using a diaphragm pump to obtain a vinyl polymer (A-1) having amine oxide groups.
The amine oxide group content of the obtained vinyl polymer (A-1) was 4.73 mmol/g based on the amount of dimethylaminopropylacrylamide added and the above-mentioned formula 1.
Next, 1 g of the obtained vinyl polymer (A-1) was added to 99 g of phosphate buffered saline (hereinafter referred to as PBS) at 25°C, stirred and dissolved, and then heated at 25°C for 24 hours. Upon standing, cell culture medium additive 1 diluted to 1% by mass with PBS was obtained.
As a result, neither separation nor precipitation of the vinyl polymer (A-1) was observed, indicating that it was completely soluble and soluble in PBS.

[Examples 6-2 to 6-12, Comparative Examples 6-1 and 6-2]
(Cell culture medium additives 2 to 14)
Vinyl polymers having amine oxide groups (A-2 to 12) and amine oxide After obtaining vinyl polymers (A-13, 14) without groups, they were dissolved in PBS to obtain cell culture medium additives 2 to 14 diluted to 1% by mass with PBS.
Note that vinyl polymers (A-13, 14) that do not have amine oxide groups did not completely dissolve when diluted to 1% by mass with PBS after removing the solvent without reacting with the oxidizing agent. Although it was shown to be insoluble in PBS, the supernatant portion was used for evaluation.

The abbreviations in Table 6-1 are shown below.
DMAPAA: Dimethylaminopropylacrylamide DEAEMA: Diethylaminoethyl methacrylate VP: Vinylpyridine VI: Vinylimidazole BA: Butyl methacrylate 2EHA: 2-Ethylhexyl acrylate ISTA: Isostearyl acrylate MMA: Methyl methacrylate St: Styrene AA: Acrylic acid HEA: Hydroxyethyl acrylate

<Evaluation of antibody-producing cell culture medium additives>
The obtained cell culture medium additive was evaluated as follows. The results are shown in Table 6-2.

[Suppression of antibody-producing cell aggregates]
Dynamis Medium (manufactured by Gibco) was used as a medium, and L-Glutamin
was added at a concentration of 1% by mass. Hereinafter, this will be referred to as the basal medium. Two sets of these were prepared, and the obtained antibody-producing cell culture medium additive was added thereto so that the concentration of the vinyl polymer (A) was 0.05% by mass or 0.5% by mass, respectively. A CHO cell line (CRL-12455 manufactured by ATCC) that secretes and produces IgG antibodies by introducing an IgG gene into two types of media with different concentrations of vinyl polymer (A) is added, and the concentration of the CHO cell line is 1,000, A cell suspension of 000 cells/ml was prepared. 20 mL of the obtained cell suspension was seeded in a 125 mL Erlenmeyer flask and cultured at 37°C. During the culture, 15 mL of the supernatant was collected once every 3 to 4 days before the nutrient source was exhausted and replaced with 15 mL of new basal medium, and this was repeated 5 times.
The ability to inhibit the formation of antibody-producing cell aggregates was evaluated by visually observing the vicinity of the liquid level in the Erlenmeyer flask after repeating the medium exchange five times.
a: Aggregates are not generated near the liquid surface of the Erlenmeyer flask: Good b: Agglomerates are slightly generated near the liquid surface of the Erlenmeyer flask: Usable c: Agglomerates are not generated near the liquid surface of the Erlenmeyer flask Uniformly occurring: Unusable

[Antibody productivity]
Cell culture was carried out in the same manner as in the evaluation of the ability to suppress the generation of antibody-producing cell aggregates, and the amount of antibody in the supernatant of the Erlenmeyer flask was measured using the Human IgG ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. after repeating the medium exchange five times. Measured using Judgment was made based on the relative value when the result when cultured without adding additives to the antibody-producing cell culture medium was set as 1.
a: 1.2 or more: Good b: 1 or more and less than 1.2: Usable c: Less than 1: Unusable

As shown in Table 6-2, the antibody-producing cell culture medium additive of the present invention containing a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 2,000 or more is added to the medium. Excellent antibody productivity was shown by adding this. It was also shown that the generation of antibody-producing cell aggregates during culture was suppressed, making it easier to purify antibodies. This is because the polarity of the vinyl polymer (A) was controlled and the environment around the antibody-producing cells was appropriately controlled, which worked effectively in terms of proliferation, survival, and antibody productivity of the antibody-producing cells. Conceivable.
On the other hand, in Comparative Examples 6-1 and 6-2 in which a vinyl polymer having no amine oxide group was used, antibody-producing cell aggregates adhered to the Erlenmeyer flask wall. Furthermore, as a result of measuring antibody productivity, the percentage of antibody amount decreased significantly. This is considered to be because the environment around the cells could not be controlled appropriately due to the absence of amine oxide groups.

<<Example of protein stabilizer>>
<Preparation of vinyl polymer (A)>
[Manufacture example 1]
(Vinyl polymer (A-1))
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen inlet tube, a reflux condenser, and a dropping tube, 150 parts by mass of ethyl acetate was charged as an organic solvent under a nitrogen stream, and the mixture was heated at 80° C. for 30 minutes with stirring. A dropping tube was charged with 80 parts by mass of dimethylaminopropylacrylamide as monomers, 20 parts by mass of butyl acrylate, 0.5 parts by mass of azobisisobutyronitrile as a polymerization initiator, and 10 parts by mass of ethyl acetate as a solvent, and left for 2 hours. It dripped. Heating was continued for 6 hours after the completion of the dropwise addition. Thereafter, the reaction was stopped by cooling to room temperature.
Next, 150 parts of ethanol and 50 parts of 35% hydrogen peroxide as an oxidizing agent (equimolar amount to the dimethylaminopropylacrylamide used) were added and reacted at 70°C for 16 hours to oxidize the tertiary amino group. I did it. Thereafter, the solvent was removed using a diaphragm pump to obtain a vinyl polymer (A-1) having amine oxide groups.
The amine oxide group content of the obtained vinyl polymer was 4.73 mmol/g based on the added amount of dimethylaminopropylacrylamide and the above-mentioned formula 1.
In addition, 1 g of the obtained vinyl polymer (A-1) was put into 99 g of ion-exchanged water at 25° C., stirred and dissolved, and then left at 25° C. for 24 hours. As a result, these resins showed no separation or precipitation, indicating that they were completely soluble and water-soluble.

[Production Examples 2 to 14]
(Vinyl polymer (A-2 to 14))
Vinyl polymers with amine oxide groups (A-2 to 12) and vinyl polymers without amine oxide groups (A-13 , 14) were obtained. Note that the vinyl polymers (A-13, 14) are those obtained by removing the solvent without reacting with the oxidizing agent.

The abbreviations in Table 7-1 are shown below.
DMAPAA: Dimethylaminopropylacrylamide DEAEMA: Diethylaminoethyl methacrylate VP: Vinylpyridine VI: Vinylimidazole BA: Butyl methacrylate 2EHA: 2-Ethylhexyl acrylate ISTA: Isostearyl acrylate MMA: Methyl methacrylate St: Styrene AA: Acrylic acid HEA: Hydroxyethyl acrylate

<Preparation of protein stabilizer aqueous solution>
[Example 7-1]
(Protein stabilizer aqueous solution (AS-1))
The obtained vinyl polymer (A-1) was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain an aqueous protein stabilizer solution (AS-1) with a solid content concentration of 0.1% by mass.

[Examples 7-2 to 7-12, Comparative Examples 7-1 to 7-2]
(Protein stabilizer aqueous solution (AS-2 to 14))
Protein stabilizer aqueous solutions (AS-2 to AS-14) were obtained in the same manner as in Example 7-1, except that the vinyl polymer (A-1) was changed to the vinyl polymer shown in Table 7-2. Ta.

[Comparative Example 7-3]
(Protein stabilizer aqueous solution (AS-15))
A 35% N,N-dimethyldodecylamine N-oxide solution (manufactured by Fuji Film Wako Chemical) was diluted with a PBS solution to obtain an aqueous protein stabilizer solution (AS-15) with a solid content concentration of 0.1% by mass. N,N-dimethyldodecylamine N-oxide is a low molecular weight compound (molecular weight 229.46) containing an amine oxide group.

<Evaluation of protein stabilizers>
The obtained protein stabilizer was evaluated as follows. The results are shown in Table 7-2.

[Preparation of enzyme aqueous solution for evaluation]
HRP-labeled CRP antibody (manufactured by ABCAM) was diluted and mixed with 1 ml of the obtained protein stabilizer aqueous solution to a concentration of 8.0 ng/ml to prepare an enzyme aqueous solution for evaluation. The enzyme aqueous solution for evaluation was tightly sealed and stored at 25° C. in a dark place so that there would be no change in concentration.

[Enzyme activity evaluation]
Using a coloring kit for peroxidase (manufactured by Sumitomo Bakelite), mix 10ml of coloring agent (3,3',5,5'-tetramethylbenzidine (TMBZ) solution) and 100μl of substrate solution (hydrogen peroxide solution) to create a coloring solution. I got it.
100 μl of this coloring solution and 100 μl of the enzyme aqueous solution for evaluation were added to a 96-well plate, and the plate was left standing in the dark at 30° C. for 20 minutes. 100 μl of a stop solution (sulfuric acid aqueous solution) was added to this to stop the reaction. The absorbance at 450 nm was measured using a plate reader Multimode Reader Mithras2 LB943 (manufactured by BERTHOLD TECHNOLOGIES) to quantify the amount of TMBZ oxide produced by the reaction. Since the oxidation reaction of TMBZ correlates with the activity of the HRP-labeled CRP antibody, the obtained absorbance can be used as an indicator of enzyme activity.

[Evaluation of protein stabilization effect]
The above enzyme activity evaluation was performed twice, before storage of the enzyme aqueous solution for evaluation and after storage at 25° C. for 30 days, and the absorbance was measured. Relative absorbance was calculated by calculating the ratio of absorbance after storage to absorbance before storage as 100%, and protein denaturation was evaluated based on the following criteria.

Relative absorbance after 30 days storage ◎: 95% or more (extremely good)
○: 90% or more, less than 95% (good)
△: 80% or more, less than 90% (usable)
×: Less than 80% (unusable)

As shown in Table 7-2, a high protein stabilizing effect was obtained by using the protein stabilizer containing the vinyl polymer having an amine oxide group of the present invention.
On the other hand, Comparative Examples 7-1 to 7-2 using a vinyl polymer without amine oxide groups and Comparative Example 7- using a low molecular weight compound having an amine oxide group but with a mass average molecular weight of less than 2,000. It was found that a sufficient stabilizing effect could not be obtained even when No. 3 was stored in coexistence with protein. This is considered to be because the protein does not have an amine oxide group, or even if it does have an amine oxide group, it has a low molecular weight, so the external environment of the protein cannot be controlled appropriately.

Claims (23)

A protein, cell or microorganism containing a vinyl polymer (A) having an amine oxide group, a mass average molecular weight of 2,000 or more, and a content of the amine oxide group of 1 mmol/g or more. An adhesion inhibitor ,
The vinyl polymer (A) is a product obtained by polymerizing a monomer having an ethylenically unsaturated group,
The monomer having an ethylenically unsaturated group is a monomer (b) having one ethylenically unsaturated group and an amine oxide group in one molecule, and a monomer (c) having an alkyl group having 1 to 18 carbon atoms. adhesion inhibitors for proteins, cells or microorganisms, including The adhesion inhibitor for proteins, cells, or microorganisms according to claim 1, wherein the vinyl polymer (A) has at least one structure represented by the following general formulas 1 to 3.



[In general formulas 1 to 3,
X is a divalent bonding group,
y is 0 or 1,
R ¹ is an alkylene group having 1 to 6 carbon atoms,
R ² and R ³ are each independently an alkyl group having 1 to 4 carbon atoms,
R ⁴ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Four of R ⁵ to R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ⁵ to R ⁹ is a bond with the main chain of the vinyl polymer (A). represents the bond position,
* represents the bonding position with the main chain of the vinyl polymer (A). ] The method for inhibiting adhesion of proteins, cells, or microorganisms according to claim 1 or 2 , wherein the vinyl polymer (A) has an amine oxide group content of 10 mmol/g or less and is used as a blocking agent for biochemical analysis. agent. The adhesion inhibitor for proteins, cells, or microorganisms according to claim 3, wherein the vinyl polymer (A) is a copolymer consisting of a thiol group-containing compound (x) as a chain transfer agent and a vinyl monomer. After bringing the adhesion inhibitor for proteins, cells, or microorganisms according to claim 3 or 4 into contact with the pathological tissue fixed on the surface of the base material, a staining antibody is adsorbed to the antigen in the pathological tissue, and the antigen An immunostaining method to detect. The adhesion inhibitor for proteins, cells, or microorganisms according to claim 1 or 2, which is used as a coating agent for inhibiting biofilm formation. The protein, cell or microorganism adhesion inhibitor according to claim 6 , wherein the vinyl polymer (A) contains 1 to 5 mmol/g of amine oxide group. A laminate for inhibiting biofilm formation, comprising a coating film comprising the protein, cell, or microorganism adhesion inhibitor according to claim 6 or 7 on a base material. The protein, cell, or microorganism adhesion inhibitor according to claim 1 or 2 , which is used in a cell culture equipment treatment agent. The protein, cell or microorganism adhesion inhibitor according to claim 9 , wherein the vinyl polymer (A) contains 1 to 5 mmol/g of amine oxide group. The protein, cell, or microorganism adhesion inhibitor according to claim 9 or 10 , wherein the vinyl polymer (A) has at least one selected from the group consisting of carboxyl groups and hydroxyl groups. The protein, cell or microorganism adhesion inhibitor according to claim 11 , further comprising a crosslinking agent. A cell culture device having, on a base material, a cell adhesion prevention film containing the protein, cell or microorganism adhesion inhibitor according to any one of claims 9 to 12 . A medical device comprising the cell culture equipment according to claim 13 . The adhesion inhibitor for proteins, cells, or microorganisms according to claim 1 or 2 , which is used as a protein stabilizer. The protein, cell or microorganism adhesion inhibitor according to claim 15, wherein the vinyl polymer (A) contains 1 to 10 mmol/g of amine oxide group. A method for stabilizing proteins, which comprises coexisting the protein, cell, or microorganism adhesion inhibitor according to claim 15 or 16 with the protein. The protein, cell, or microorganism adhesion inhibitor according to claim 1 or 2 , which is used as a cell culture medium additive. The protein, cell, or microorganism adhesion inhibitor according to claim 1 or 2 , which is used as an antibody-producing cell culture medium additive. The protein, cell or microorganism adhesion inhibitor according to claim 18 or 19 , wherein the vinyl polymer (A) contains 1 to 10 mmol/g of amine oxide group. A cell culture medium comprising the protein, cell or microorganism adhesion inhibitor according to any one of claims 18 to 20 . The cell culture medium according to claim 21 , wherein the concentration of the vinyl polymer (A) in the cell culture medium is 0.01% by mass or more and 1% by mass or less. A cell culture method comprising culturing cells in the cell culture medium according to claim 21 or 22 .